Flow Cytofluorometry in Transfusion Medicine


Vol. 15 •Issue 8 • Page 13
The Learning Scope:

Flow Cytofluorometry in Transfusion Medicine — 0.1 CEU.or 1 Contact Hour

Flow Cytofluorometry in Transfusion Medicine

As flow cytofluorometers become more available in clinical laboratories, transfusion medicine scientists will want to take advantage of them.

This offering expires April 7, 2004

Editor’s note: This Learning Scope article offers a unique opportunity for clinical laboratory personnel to earn continuing education credit. It is offered to our subscribers six times a year. Simply read the peer-reviewed article and send your answers to the questions that follow to the American Society for Clinical Laboratory Science (ASCLS). As does this one, most Learning Scope articles provide 0.1 CEU or 1 contact hour if you score 70 or above. The credit is issued through ASCLS’ PACE program. The ASCLS is the oldest and largest non-registry, non-profit professional membership association representing non-physician clinical laboratory practitioners. You don’t need to be a member to earn the credit. Additional details concerning the CEU offer are listed to the left of the answer sheet on page 19. Tests may be submitted up to one year following publication in ADVANCE. The answer sheet may be copied for a fellow laboratorian to submit for credit.

Learning Objectives

At the conclusion of this article the participant will be able to:

• Name the four main components of a flow cytofluorometer.

• Describe five ways to avoid RBC agglutination.

• List eight uses of flow cytofluorometry in immunohematology.

• Identify two methods for detecting and quantitating a fetal-maternal hemorrhage by flow cytometry.

Flow cytometers are instruments that make physical and/or chemical measurements on individual cells in a fluid stream, and include instruments used for complete blood counts in the clinical laboratory (e.g., a Coulter counter). Flow cytometers that measure cellular fluorescence are called flow cytofluorometers. These instruments make measurements on thousands of cells as they pass single file in a fluid stream through a focused beam of light, usually a laser beam.

Flow cytofluorometers are found not only in research laboratories but also in clinical laboratories where their uses include diagnosis, prognosis and minimal disease detection in leukemia/lymphoma, monitoring the progression of HIV and quantification of stem cells for transplantation. Other clinical uses include analysis of DNA ploidy and cell division kinetics, quantitation/analysis of mature and immature reticulocytes and diagnosis of paroxysmal nocturnal hemoglobinuria.

Flow cytofluorometry (FC) also has been found to have uses in transfusion medicine (Table 11,2), which will be discussed here, along with principles of the science.

Principles of Flow Cytofluorometry

A flow cytofluorometer consists of four main components:

1. The fluidics (or flow) system that, through hydrodynamic focusing, positions cells so that they pass, individually, through a beam of light;

2. The optical system, consisting of the light source (e.g., an argon ion laser), the lenses that direct the light beam to the observation point where the cells are illuminated, and the lenses and filters that collect and/or separate light or fluorescence that has been scattered in a forward direction (forward scatter) or at a 90º angle (side scatter) and direct it to the appropriate detectors (photodiodes or photomultiplier tubes);

3. The electronic system that transforms the light information to an analog format (i.e., signal pulse height, area or width) and then to a digital format; and

4. The computer system, used to store and analyze the data that is generated by the cells as they pass through the light beam. Data are commonly represented as single parameter histograms or dual parameter dot plots (e.g., forward scatter versus side scatter; green fluorescence versus orange-red fluorescence). Results are usually expressed as the percentage of positive cells (relative to a negative control) or as a measure of fluorescence intensity (e.g., mean or median fluorescence).

Information from scattered light can give relative information about a cell’s size and its complexity and can be used, for example, to distinguish different white cell subtypes (e.g., lymphocytes, monocytes, granulocytes). One useful feature of FC is that an electronic “gate” or “region” can be set around a subpopulation of interest to obtain information about the subpopulation without having to perform a physical separation.

Use of Fluorescent Dyes

Fluorochromes (fluorescent dyes) are used to label cellular components. Flow cytofluorometers with one laser (e.g., an argon ion laser) can typically measure up to three or four colors of fluorescence. Those with multiple lasers can measure many more colors. Cells can be directly stained with fluorochromes such as propidium iodide or thiazole orange (which are used as DNA and RNA labels, respectively). For studying external surfaces of cells such as cell surface antigens, fluorochrome-labeled antibodies or lectins are used.

The most commonly used fluorochromes are fluorescein isothiocyanate (FITC) and phycoerythrin (PE), both of which are excited by the blue light of an argon ion laser and fluoresce green and orange-red, respectively. Cells (e.g., RBCs) can be labeled directly with a fluorochrome-labeled primary antibody (e.g., FITC anti-D) or indirectly with fluorochrome-labeled secondary antibody (e.g., anti-D + FITC anti-IgG, or anti-D + biotin anti-IgG + avidin-FITC). If cells are fixed and permeabilized, then intracellular antigens (e.g., fetal hemoglobin) can be labeled using fluorochrome-labeled antibodies.

Agglutination Can Present Problem

A technical problem that can affect RBC analysis by FC is the presence of agglutination. An agglutinated cluster of RBCs may not only block the flow cell but will be counted erroneously by the flow cytofluorometer as one very large, possibly very fluorescent cell. This could adversely affect the interpretation of results (e.g., when quantitating a subpopulation of cells). Thus, agglutination is to be avoided and there are different ways of accomplishing this, including diluting antisera so that it no longer agglutinates, pretreating IgM antibodies with sulfhydryl reagents, avoiding potentiators (e.g., albumin), pretreating RBCs with a chemical like glutaraldehyde, or using fluorescein-labeled antibody fragments (e.g., Fab FITC anti-IgG). Mechanical methods (e.g., passing agglutinated samples through a fine gauge needle or small-bore pipette) have been used to break up agglutinates, but these methods are not always effective and may cause hemolysis of RBCs.

Applications in Blood Transfusion Science

Table 1 summarizes the major uses of FC in blood transfusion science. The applications to RBC immunohematology will be the focus here, but the principles are similar for platelet and white cell antigens and antibodies.

Detection and quantitation of RBC-bound proteins (e.g., IgG, IgG subclasses, IgM, IgA, C3)–The antiglobulin test (AGT) is a simple, cheap and sensitive method, with an ability to detect about 100-200 IgG molecules per RBC. FC is usually no more sensitive than this, but in special situations it appears to detect RBC-bound IgG or IgM when the AGT fails (i.e., the direct antiglobulin test [DAT] is negative). RBC-bound IgM can be particularly difficult to detect by the DAT, but is easily detected by FC where sensitization, not agglutination, is the endpoint.

FC can be used to quantitate RBC-bound immunoglobulins. Garratty and Nance3 showed that D+ RBCs sensitized with different amounts of anti-D, so that the RBCs all reacted strongly by routine (test tube) AGT (4+), could be differentiated using FITC anti-IgG and FC (mean fluorescence results of the 1 in 5, 1 in 10, 1 in 20 and 1 in 40 dilutions of anti-D were 212.0, 183.2, 130.1 and 84.7, respectively). Thus, FC has the advantage over serology of making quantitative differences clearer. The number of molecules of immunoglobulin (Ig) per RBC can be calculated from a standard curve using Ig-coated beads of known concentrations.

Van der Meulen et al4 used FC to determine if a quantitative relationship existed between the amount of IgG1 autoantibodies on RBCs from patients with a positive DAT and the presence or absence of hemolytic anemia in those same patients. Although these investigators found a quantitative threshold for IgG-mediated in vivo RBC destruction,4 this could not be confirmed by Garratty and Nance3 when a larger group of patients was studied.

Detection and quantitation of RBC antigens–FC has been used to study a number of blood group antigens, including ABH and Rh. Studying ABH antigens on RBCs can be technically difficult due to the strong agglutination caused by anti-A and anti-B. Agglutination by anti-A or anti-B can be avoided by chemically pretreating the RBCs and/or it can be dispersed by mechanical means (see earlier). Fibach and Sharon5 used FC to show that ABH antigen density decreases as RBCs age in vivo.

FC has been used to show the variation in antigen strength on RBCs of differing phenotypes (e.g, increasing D antigen strength on DCcee [R1r], DccEE [R2R2], and the rare -D- RBCs), and for quantitating antigens on RBCs. Oien et al6 showed that FC is superior to manual titration scores when determining zygosity. In the 1980s, FC was found to be useful in paternity testing (i.e., differentiating RBCs from heterozygotes and homozygotes) but paternity testing is now performed by genetic methods (some of these methods use flow cytofluorometers).

Detection and quantitation of RBC antibodies–FC can be used to quantitate antibody in serum. The serum antibody is incubated with antigen positive RBCs and the amount of IgG on the RBCs is then determined by the addition of a fluorochrome-labeled anti-IgG and FC (same as when studying antigens). As previously mentioned, when D+ RBCs are incubated with different amounts of anti-D, then FITC anti-IgG and analyzed by FC, a clear quantitative difference in the amount of anti-D on the cells (and thus the amount of anti-D in the serum) can be seen.

In pregnant women with anti-D, the amount of anti-D is determined in the United States by titration using the AGT. The result of this testing (e.g., titer) determines whether a woman should have an invasive test (e.g., amniocentesis) to assess if the fetus is affected by hemolytic disease of the newborn. In Europe, the amount of anti-D in prenatal maternal serum is quantitated in micrograms or international units (IU) using an AutoAnalyzer method and a standard anti-D curve. More recently, Europeans have been evaluating FC methods as alternatives to the AutoAnalyzer method.7

Detection and quantitation of minor RBC populations–One advantage of FC is that it is an efficient method for studying minor cell populations. Because each cell is analyzed individually, those with different levels of fluorescence can be differentiated and quantitated. There are a number of applications:

• Fetal-maternal hemorrhage detection and quantitation–It is important to be able to detect and quantitate a fetal-maternal hemorrhage (FMH) of greater than 15 mL (~ 0.6 percent) fetal blood so that the appropriate dose of Rh immune globulin (RhIG) can be administered (in the United States, one 300 µg dose covers a 15 mL fetal bleed). If too small a dose is given, the woman may make anti-D, which could affect a future pregnancy. RhIG is in short supply so giving too large a dose should also be avoided.

The rosetting method is a sensitive means for detecting an FMH but a quantitative method must then be used. The commonly used Kleihauer-Betke acid elution method for quantitating an FMH is subjective and has poor reproducibility. A number of papers have described methods using FC to quantitate FMH. The methods that have been used involve labeling fetal D+ cells with a fluorescent-labeled anti-D by either a direct8 or indirect method9, or labeling fetal hemoglobin with FITC anti-HbF.10 The anti-D method is only useful when there is a D antigen difference between the mother and fetus.

In contrast, the anti-HbF method could be used in quantitating a fetal bleed in a trauma case where there may not be a difference in D antigen typing between the mother and fetus. The anti-HbF method requires that the RBCs be permeabilized (using glutaradehyde and Triton X-100) so that the antibody can have access to the intracellular HbF, and is commercially available as a kit.

Both the anti-D and anti-HbF methods of FMH quantitation have been shown to have good sensitivity, precision and accuracy. The correlation with Kleihauer-Betke results also have been good, although it has been noted in several publications that FC results by the anti-D method tend to be lower than acid elution results. Hereditary persistence of HbF in adult cells can be the cause of falsely elevated results by the acid elution methods, but the FC anti-HbF method can differentiate these cells from fetal RBCs (the adult RBCs with hereditary persistence of HbF are less fluorescent than fetal cells).

• Detection and quantitation of genetic chimerism and mosaicism—Some rare individuals have two populations of cells. Genetic chimeras are twins who shared blood-forming tissue in utero. Cellular mosaicism can occur when a somatic mutation occurs in a stem cell (e.g., the Tn antigen) or in carrier females of X-linked genes (e.g., the Xk locus, which is associated with weak expression of Kell antigens [McLeod phenotype]) due to the Lyon phenomenon of X chromosome inactivation. By FC, the two populations can be clearly differentiated and quantitated. One chimeric blood donor whose RBCs showed mixed field agglutination with anti-B could be shown, using FC, to have a minor population (35 percent) of B, Fy(a-), Jk(b-) RBCs and a major population (65 percent) of O, Fy(a+), Jk(b+) RBCs.1 An FC study on RBCs from 12 female carriers of the McLeod phenotype showed the proportion of RBCs with depressed Kell antigens to vary from 8-82 percent in the different women.11

• Detection and quantitation of transfused RBCs (RBC survival studies)–FC has been used to follow the survival of a transfused unit of blood. Usually this involves following an antigen that is present on the transfused cells and not present on the recipient’s cells, although the opposite approach has also been used. Samples are collected from the patient pre-transfusion and at different times post-transfusion and the percentage of transfused cells is determined. The sensitivity of the assay depends upon the strength of the antigen and antibody that are being studied; not all antigens are good choices to be studied. The FC approach has been used successfully for RBC survival studies in patients with anti-Jka, -Jsb, -Dib and -B. In the last case, two-color fluorescence was used to differentiate RBCs with acquired B antigen from RBCs with genetically determined B antigen, and to follow the survival of these RBCs in a patient suffering from an ABO hemolytic transfusion reaction.12

FC also has been used to follow the survival of a small aliquot of transfused RBCs (e.g., 10 mL), similar to survival studies performed with 51Cr-labeled aliquots of RBCs (1 mL). These FC studies have been performed successfully in patients with anti-Lu6 (anti-D was used to follow the major population of cells) and anti-Ge. Read et al13 studied RBCs labeled with PKH2, a fluorescent dye that binds to cell membranes, and found that they could detect 0.01 percent labeled RBCs in in vitro experiments.

• Phenotyping recipient’s RBCs (reticulocytes) following transfusion—It is difficult to determine a patient’s true phenotype when transfused RBCs are present. Most approaches involve physical separation of younger (recipient’s) RBCs from older (transfused and recipient’s) RBCs using centrifugation with and without density gradients. These methods are labor intensive, subjective, not reliable and require a significant amount of sample. In 1994, Griffin et al14 described a two-color method using FC to phenotype recipient RBCs following transfusion. Thiazole orange (a green fluorochrome) was used to label reticulocytes and phycoerythrin (an orange-red fluorochrome) was used in an indirect method to label the antigen of interest. Antigen positive reticulocytes would fluoresce red and green and antigen negative reticulocytes would fluoresce only green; no physical separation of cells is necessary. Their in vitro and in vivo data showed that this method could be performed successfully with most antibodies and their limit of detection was 0.3 percent reticulocytes. As with any of the methods used to type reticulocytes, it is important to wait 48-72 hours after transfusion to perform the typing, so that the transfused reticulocytes have time to mature.

• Detection and quantitation of chimerism associated with bone marrow transplantation—There are a number of methods that have been used to follow bone marrow transplant (BMT) engraftment. Methods that involve DNA are preferred because they are not affected by transfusion, but agglutination and FC methods have been used to follow the disappearance and appearance of RBC antigens on the recipient’s and transplanted donor’s cells, respectively. Single-color and two-color (using thiazole orange to label reticulocytes), direct and indirect FC methods have been used by different investigators. A sensitivity level of 0.1 percent could be reached using a two-color indirect method.

FC was found to be particularly useful in differentiating the presence of a relapse in a group A1 patient, who had received an O bone marrow transplant, from the phenomenon of soluble plasma antigen uptake.15 This phenomenon of uptake of A or B plasma antigens onto O RBCs has been noted in A and B recipients of O transfusions or BMTs and in ABO chimeras. The amount of A or B substance in plasma is affected by the patient’s ABO, Se (secretor) and Le (Lewis) genes.

The patient in question typed as group O 1 month post-transplant, but 3-4 months later the patient’s RBCs were reacting weakly with some monoclonal and polyclonal anti-A and anti-A,B by tube and gel methods. FC was used to determine if the patient was having a relapse (i.e., a chimera) or if soluble A antigen had been taken up by his O RBCs and was being detected. The patient’s RBCs were incubated with anti-A and FITC anti-Ig. If the patient were having a relapse, then a small population of brightly fluorescent A cells would have been detected. If the transplanted O cells had taken up small amounts of soluble plasma A antigen, then all cells would be shown to be weakly fluorescent (similar to what is seen when testing weak A subgroup RBCs). FC testing showed weak fluorescence of this patient’s RBCs, thus confirming uptake of A antigen onto the transplanted O RBCs.

Advantages and Disadvantages

Flow cytofluorometers have been found to be extremely useful in many fields of study because they can reproducibly, objectively and quantitatively measure multiple characteristics of large numbers of cells quickly. The generation of data from several thousands of cells in less that 1 minute allows for increased statistical precision and accuracy. FC is especially useful in the analysis of mixed populations (e.g., rare event analysis) because each cell is analyzed individually and subpopulations can be studied without having to physically separate cells. A small sample size is needed for most analyses and FC is a more sensitive method than fluorescence microscopy. Results of FC analyses can be stored permanently in computer files to be reanalyzed at a later date.

The disadvantages of FC are that the instrument and the fluorescent reagents are expensive. Reagents, even those sold for FC, may not have been standardized by FC and the user will need to be aware of this and perform the needed standardization. In addition, reagents are not intended for use with RBCs. Individual cells are required for analysis (thus no agglutinates). Although thousands of cells can be analyzed by a flow cytofluorometer in less than 1 minute, the increased set-up and data analysis times can sometimes result in this method taking longer than other methods (e.g., agglutination).

Conclusion

In conclusion, although for many of the tests performed in immunohematology the method of choice is agglutination, which is a quick, simple and low-cost method, agglutination methods will not always give us needed information. In many of those cases, FC can be a useful technology. Most of this work has been performed in research labs rather than clinical labs, but as flow cytofluorometers are becoming more available in clinical laboratories, hopefully transfusion medicine scientists will be able to take advantage of them.

References

1. Garratty G, Arndt P. Applications of flow cytofluorometry to transfusion science. Transfusion 1995;35:157-178.

2. Garratty G, Arndt P. Applications of flow cytofluorometry to red blood cell immunology. Cytometry (Communications in Clinical Cytometry) 1999;38:259-267.

3. Garratty G, Nance SJ. Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry. Transfusion 1990;30:617-621.

4. van der Meulen FW, de Bruin HG, Goosen PCM, et al. Quantitative aspects of the destruction of red cells sensitized with IgG1 autoantibodies: An application of flow cytometry. Br J Haematol 1980;46:47-56.

5. Fibach E, Sharon R. Changes in ABH antigen expression on red cells during in vivo aging: A flow cytometric analysis. Transfusion 1994;34:328-32.

6. Oien L, Nance S, Arndt P, Garratty G. Determination of zygosity using flow cytometric analysis of red cell antigen strength. Transfusion 1988;28:541-4.

7. Austin EB, McIntosh Y. Anti-D quantification by flow cytometry: A comparison of five methods. Transfusion 2000;40:77-83.

8. Lloyd-Evans P, Kumpel BM, Bromilow I, et al. Use of a directly conjugated monoclonal anti-D (BRAD-3) for quantification of fetomaternal hemorrhage by flow cytometry. Transfusion 1996;36:432-7.

9. Nance SJ, Nelson JM, Arndt PA, et al. Quantitation of fetal-maternal hemorrhage by flow cytometry, a simple and accurate method. Am J Clin Pathol 1989;91:288-92.

10. Davis BH, Olsen S, Bigelow NC, Chen JC. Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry. Transfusion 1998;38:749-56.

11. Reid ME, Oyen R, Ralph H, Rubinstein P. Demonstration of McLeod phenotype RBCs. Transfusion 1994;34:21S. (abstract)

12. Garratty G, Arndt P, Co A, et al. Fatal hemolytic transfusion reaction resulting from ABO mistyping of a patient with acquired B antigen detectable only by some monoclonal anti-B reagents. Transfusion 1996;36:351-7.

13. Read EJ, Cardine LL, Yu MY. Flow cytometric detection of human red cells labeled with a fluorescent membrane label: Potential application of in vivo survival studies. Transfusion 1991;31:502-8.

14. Griffin GD, Lippert LE, Dow NS, et al. A flow cytometric method for phenotyping recipient red cells following transfusion. Transfusion 1994;34:233-7.

15. Garratty G, Arndt PA, Noguerol P, Plaza E, Jiminez J. Differentiation of post-bone marrow transplant chimerism versus adsorption of A antigen onto transplanted group O RBCs. Transfusion 1999;39:44S. (abstract)

Patricia Arndt is senior research associate, American Red Cross, Southern California Region, Los Angeles.

Table 1: Applications of Flow Cytofluorometry to Blood Transfusion Science

• Detection and quantitation of

– RBC- and platelet-bound proteins (e.g., IgG, C3)

– antigens on RBCs and platelets

– RBC, WBC and platelet antibodies

• Detection of minor cell populations or rare events

– RBCs (e.g., fetal maternal hemorrhage, transfusion, bone marrow transplant)

– WBCs (leukoreduced products)

• Platelet function

table, figures/courtesy Patricia Arndt, MS, MT(ASCP)SBB

Learning Scope Questions

1. A flow cytometer can most likely be found in which clinical laboratory department?

a) General Chemistry

b) Hematology

c) Microbiology

d) Transfusion Service

2. The clinical uses of flow cytofluorometry include all the following except:

a) quantitation of immature reticulocytes.

b) diagnosis of leukemia/lymphoma.

c) diagnosis of paroxysmal cold hemoglobinuria.

d) diagnosis of paroxysmal noctural hemoglobinuria.

3. The four main components of a flow cytofluorometer include all except which of the following:

a) Fluidics system

b) Computer

c) Electronic system

d) Sorter

4. The optical system of a flow cytofluorometer consists of all except which of the following:

a) Photodiodes and photomultiplier tubes

b) Light source

c) Cuvette

d) Lenses and filters

5. Which of the following statements is false?

a) Light scatter gives information about a cell’s size and complexity.

b) Flow cytofluorometers with a single laser can measure six colors of fluorescence.

c) A “gate” can be used to electronically separate a subpopulation of cells.

d) Results are commonly expressed as % positive or mean/median fluorescence.

6. Which of the following is a fluorochrome that will directly stain cells?

a) Thiazole orange

b) Phycoerythrin

c) Biotin/avidin

d) Fluorescein isothiocyanate

7. What treatment is needed to label internal antigens with fluorochrome-labeled antibodies?

a) Pretreatment of RBCs with glutaraldehyde

b) Dilute antibodies

c) Pretreatment of antibody with sulfhydryl reagents

d) Fixation/permeabilization of RBCs

8. Analysis of agglutinated RBCs by flow cytofluorometry can cause all of the following problems except:

a) carryover from one sample to the next.

b) blocking of the flow cell.

c) false elevation of the mean/median fluorescence results.

d) erroneous quantitation of a subpopulation of cells.

9. The antiglobulin test has a sensitivity of:

a) 20-50 IgG molecules/RBC.

b) 100-200 IgG molecules/RBC.

c) 300-400 IgG molecules/RBC.

d) >500 IgG molecules/RBC.

10. Which of the following statements regarding the ability of flow cytofluorometry to quantitate antibody on RBCs and in serum is true?

a) Flow cytofluorometry makes quantitative differences less clear than the antiglobulin test.

b) Flow cytofluorometry and the AGT are similar in their ability to make quantitative differences clear.

c) Flow cytofluorometry makes quantitative differences more clear than the antiglobulin test.

d) Flow cytofluorometry cannot be used to evaluate quantitative differences.

11. With regards to fetal-maternal hemorrhage prophylaxis, one 300 mg dose of RhIG will cover what size fetal bleed (packed cells)?

a) 5 mL

b) 10 mL

c) 15 mL

d) 20 mL

12. Cellular mosaicism occurs when:

a) twins share blood-forming tissue in utero.

b) there is a somatic mutation in a stem cell.

c) a relapse occurs after a bone marrow transplant.

d) a patient is transfused allogeneic blood.

13. Using flow cytofluorometry to determine the antigen status of reticulocytes (i.e., thiazole orange to label reticulocyte RNA and phycoerythrin to label the antigen of interest), which fluorescent color(s) would antigen positive reticulocytes emit?

a) Red only

b) Green only

c) Red and green

d) None

14. The amount of A or B substance in the plasma of a group A or B person depends upon all the following except:

a) ABO gene

b) Se gene

c) H gene

d) Le gene

15. Advantages of flow cytofluorometry include all of the following except:

a) increased statistical precision and accuracy.

b) sensitivity.

c) objectivity.

d) cost.