Flow cytometric testing in hematology – Why is it relevant to every clinical laboratory today?

Abstract

AIM: To highlight the efficacy of flow cytometry in the diagnosis, classification, prognostic evaluation and detection of minimal residual disease in leukemias, lymphomas and immunologic monitoring of HIV- infected patients.

METHODOLOGY: Relevant scientific papers was retrieved to pinpoint the principles of operation of flow cytometry, clinical applications, sources of error and important steps towards definitive and specific diagnosis.

CONCLUSION: Flow cytometry is relevant to every clinical laboratory today because of its application in diagnosis of various hematological malignancies and its role in clearly defining the complexity of hematopoietic disorders according to lineage and maturation stages with the aid of differentiation markers. It is safe, rapid, sensitive, specific and user friendly.

KEYWORDS: Flow cytometry, Hematology, Relevant, Clinical laboratory

Introduction

Flow cytometry provides rapid analysis of multiple characteristics of single cells. The information obtained is both qualitative and quantitative. However, in the past flow cytometers were found only in larger Hospitals and academic centres but advances in technology now makes it possible for community Hospitals to use this methodology. Modern flow cytometers are much smaller, less expensive, more user friendly and well suited for high volume operation.

General Principles

Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei, microorganisms, chromosome preparations and proteins). Physical properties such as size (represented by forward angle light scatter )and internal complexity (represented by light angle scatter) can resolve certain cell populations. Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes each with similar excitation wavelengths and different emission wavelengths (or colors) allows several cell properties to be measured simultaneously. Commonly used dyes include propidium iodide, phycoerythrin and fluorescein although many other dyes are now available.

Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid that creates laminar flow, allowing the cells to pass individually through an interrogation point. At the interrogation point , a beam of monochromatic light, usually from a laser, intersects the cells. Emitted light is given off in all directions and is collected via optics that direct the light to series of filters and dichroic mirrors that isolate particular wavelength bands. The light signals are detected by photo multiplier tubes and digitized for computer analysis. The resulting information usually is displayed in histogram or two dimensional dot- plot formats. FC has undergone major technical advances to provide high sensitivity and specificity through improved instrumentation, introduction of new monoclonal antibodies, utilization of up to 6 color staining, permeabilizing agents and CD45 gating strategies (Olaniyi 2011)

Pre- analytical factors for blood and bone marrow samples are that;

• 1. Anticoagulant (EDTA or Heparin) may be used

• 2. The specimen is analyzed within 24 hours

• 3. Storage should be at 2oc to 20oc

• 4. There is no need to isolate mononuclear cells

• 5. Great care be taken with lysing procedures.

It is recommended that immunophenotyping should be assessed in conjunction with clinical features and cell morphology with strict adherence to quality system essentials and careful selection of fluorochromes, antibodies whose normal expression is rather dim for example (CD7, CD10, CD116, CD13, CD2, CD34, CD64, CD117, or Tdt) should be labeled with the brightest fluorochromes like Phycoerythrin and if instrumentation allows, allophycocyanin.

Extensive research have been done on the factors that affect the results of immunophenotyping and this includes;

1.The type and quality of samples

2.The reagent and sample preparation protocols.

3.Instruments set up and calibration

4.Potential components of subjectivity introduced during data analysis or with the interpretation of the results represent the most common sources of variability.

FC is used for immunophenotyping of a variety of specimens, including whole blood, bone marrow, serous cavity fluids, cerebrospinal fluid, urine and solid tissues. Characteristics that can be measured include cell size, cytoplasmic complexity, DNA or RNA content and a wide range of membrane bound and intracellular proteins.

WHY IS FLOW CYTOMETRY RELEVANT TODAY?

The relevance of FC will be discussed under the following heading;

1.Erythrocyte Analysis

2.Leukocyte Analysis

3.Platelet Analysis

Immunophenotyping applications in hematology

The distribution nature of the hematopoietic system makes it amenable to flow cytometric analysis. The availability of monoclonal antibodies directed against these surface proteins permits flow cytometric analysis of erythrocytes, leukocytes and platelets. Antibodies against intracellular proteins such as myeloperoxidase and terminal deoxynucleotidyl transferase (Tdt) are also commercially available and permit analysis of an increasing number of intracellular markers.

Erythrocyte Analysis

The use of FC for the detection and quantification of fetal red cells in maternal blood has increased, currently in Cape Verde, RhD- negative women receive prophylactic Rh immune globulin at 28 weeks and also within 72 hours after birth. If feto maternal hemorrhage is suspected, the mother´s blood is tested for the presence and the quantity of fetal red cells and an appropriate amount of Rh- immune globulin is administered. The quantitative test mostly employed is the kleihauer- Betke acid elution test. This test is fraught with interobserver and interlaboratory variability and is tedious and time consuming Polesky & Sebring (1981). Now, the use of FC for the detection of fetal cells is much more objective, reproducible and sensitive than the Kleihauer – Betke test Davis et al (1998). Fluorescently labeled antibodies to the rhesus- D antigen can be used, or antibodies directed against hemoglobin F (HbF) Campbell et al (1999). This intracellular approach, which uses permeabilization of the red cell membrane and an antibody to the ? chain of human hemoglobin is precise and sensitive Davis et al (1998). This method has the ability to distinguish fetal cells from F- cells (adult cells with small amounts of hemoglobin F). However, The FC method is technically superior to the Kleihauer- Betke test, cost, instrument availability and stat access may limit its practical utility.

Paroxysmal Nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder that leads to intravascular hemolysis with associated thrombotic and infectious complications. PNH can arise in the setting of aplastic anemia and could be followed by acute leukemia Fores et al (1995). The disease is caused by deficient biosynthesis of a glycosylphosphatidyl inositol linker that anchors several complement and immunoregulatory surface proteins on erythrocytes, monocytes, Neutrophils, lymphocytes and platelets Hall & Rosse (1996). On erythrocytes, deficiencies of decay accelerating factor and membrane inhibitor of reactive lysis render red cells suspectible to complement mediated lysis Rotoli & Boccuni (1995). We know that conventional laboratory tests for the diagnosis of PNH include the sugar water test and the Ham´s test acid hemolysis test Vishnu Reddy (2007), Problems associated with these tests include stringent specimen requirements and limited specificity. Antibodies to CD55 and CD59 are specific for decay accelerating factor and membrane inhibitor of reactive lysis, respectively and can be analyzed by flow cytometry to make a definitive diagnosis of PNH Alfinito et al (1996). In affected patients, two or more populations of erythrocytes can be readily identified with different degrees of expression of CD55 and CD59.

Reticulocyte counts are based on identification of residual ribosomes and RNA in immature non nucleated red blood cells(RBCs). Practically, a blood smear is stained with a dye that precipitates the nucleic acid and the cells are counted manually Hutchison & Davey (1996). This method is subjective, imprecise, labor intensive and tedious. The Flow cytometric enumeration of reticulocytes uses fluorescent dyes that binds the residual RNA, such as thiazole orange. The FC method provides excellent discrimination between reticulocytes and mature RBCs with greater precision, sensitivity and reproducibility than the manual method. However, Howell- Jolly bodies ( a remnant of nuclear DNA) are not distinguished from reticulocytes Corash et al (1988). Because the fluorescence intensity is directly proportional to the amount of RNA and related to the immaturity of the RBC, a reticulocytes maturity index has been used clinically to assess bone marrow engraftment and erythropoietic activity and to help classify anemias. Currently some automated cell counters use similar technology to estimate reticulocytes counts Kickler T S (1999).

In the blood bank, flow cytometry can be used as a complementary or replacement test for red cell immunology, including RBC bound immunoglobulins and red cell antigens Garratty & Arndt (1999). In multiple transfused patients, determining the recipient´s blood type can be very difficult. Flow cytometry has been used to accurately identify and phenotype the recipient´s red cells Griffin et al (1994). Flow cytometry is being used increasingly in the blood bank to assess leukocyte contamination in leukocyte reduced blood products.

Leukocyte Analysis

Immunologic monitoring of HIV- infected patients is a mainstay of the clinical flow cytometry laboratory. HIV infects helper / inducer T lymphocytes via the CD4 antigen. Infected lymphocytes may be lysed when new virions are released or may be removed by the cellular immune system. As the HIV disease progresses, CD4 –Positive T lymphocytes decreases in total number. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging and monitoring disease progression and response to treatment in HIV infected persons. Quantitative viral load testing is a complementary test for clinical monitoring of disease and is correlated inversely to CD4 counts Saag et al (1996). However, CD4 counts directly assess the patients immune status and not just the amount of virus. It is likely that both CD4 T cell enumeration and HIV viral load will continue to be used for diagnosis ,prognosis and therapeutic management of HIV infected persons.

However, the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemia and lymphomas. Immunophenotyping as part of the diagnostic work up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics along with new antibodies and gating strategies has substantially enhanced the utility of FC in the diagnosis of leukemia and lymphomas. FC is very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias. Additionally, CD 45 / side scatter gating often can better isolate the blast population for more definitive phenotyping than is possible with forward scatter /side scatter gating. Although most acute myeloid leukemias are difficult to classify by phenotyping alone, FC can be useful in distinguishing certain acute myeloid leukemias such as acute promyelocytic leukemia Erber et al (1994). Flow cytometry (FC) can also be used to identify leukemias that may be resistant to therapy Maslak et al (1994)

The B cell lymphoproliferative disorders often have specific antigen patterns. The use of a wide range of antibodies allows clinicians to make specific diagnosis based on patterns of antigen expression. FC is particularly good at identifying clonality in B cell populations. Although T cells neoplasms may exhibit a predominance of antigens CD4 or CD8 but these antigens should not be considered as surrogate markers of clonality. Antigen deletions are common in T-cell lymphomas and may suggest neoplasia, but the only way to definitively diagnosis T cell clonality is by molecular methods. Flow cytometry can be used for lymphoma phenotyping of fine needle aspirates and is a powerful adjunct to cytologic diagnosis. The high sensitivity and capacity for simultaneous analysis of multiple characteristics make flow cytometry useful for the detection of minimal residual disease especially if abnormal patterns of antigen expression are present. FC is not recommended for the diagnosis of Hodgkin´s lymphoma, chronic myelogenous leukemia or myelodysplastic syndrome, although disease progression in the latter two conditions can often be monitored.

Platelet Analysis

The analysis of platelets by FC is becoming more common in both research and clinical laboratories. Platelet associated immunoglobulin assays by FC can be direct or indirect assays , similar to other platelet associated immunoglobulin immunoassays. In autoimmune thrombocytopenic Purpura, free serum antibodies are not found as frequently as platelet bound antibodies- Stockelberg et al (1996). However, FC is an excellent method for direct analysis of platelet bound antibodies, apoptosis and platelet aggregation, it has also been shown to be of benefit in detection of free plasma antibodies Gonzalez et al (1996). There are a number of situations in which one might expect platelet activation markers to demonstrate clinical utility Alberto Orfao et al(1995). These can be broadly classified as those areas involving biocompatibility issues, platelet storage and transfusion, those involving assessment of cardiovascular disease and assessment of platelet dysfunction.

Data are currently available to show that:

1.Platelets become activated during storage under blood bank conditions and activated platelets do not remain in the patient´s circulation.

2.Platelets become activated during extracorporeal circulation such as cardiopulmonary bypass and renal dialysis.

3. Platelets become activated during vascular damage, myocardial infarction and thrombosis

4. There is increased activation in patients at high risk for cardiovascular events.

The recent availability of immunological markers of platelet activation such as CD62, CD63 and CD69, combined with the unique capability of flow cytometry to identify small subpopulations of platelets and to quantitate membrane glycoprotein levels, now allows us to assess platelet function in an entirely new perspective.

The use of thiazole orange a fluorescent dye that binds RNA, allows immature platelets also called reticulated platelets to be quantified Rinder et al (1993). The reticulated platelet count can be used to determine the rate of thrombopoiesis. This measurement can separate unexplained thrombocytopenias into those with increased destruction and those with defects in platelet production.

Conclusion

Flow cytometry testing of hematological malignancies has become indispensable in view of its role in clearly defining the complex hematopoietic malignancies according to lineage and maturation stage, using differentiation markers. It is rapid, sensitive and specific. It appears expensive to maintain but it can be afforded, when the appropriate management mechanism is in place.

Reference

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Autor:

Peter Ubah Okeke

Student, School of Science & Engineering,

Atlantic International University Hawaii (www.aiu.edu)

SEPTEMBER, 2011