Leukemia Lymphoma Typing
Leukemia and Lymphoma typing is the most common clinical cytometry application in use today. Most leukemia and lymphomas can be categorized by abnormal cell populations in the marrow and blood. Cytometry can be used to differentiate leukemia's based on their abnormal antigen expression on a target population.
Single Platform CD34 StemCell Enumeration Assay
Since re infusion of purified CD34 antigen positive cells results in the hematopoitic recovery following myeloablative therapy, quantitation of cells bearing this cell surface molecule provides a rapid means to measure autograft potential in bone marrow, cord blood and mobilize peripheral blood. By using multiparameter flow cytometry methodologies certified buy the International Society of Hematology and Graft Engineering (ISHAGE) we are able to measure this rare CD34 population with accurate and reproducible results. With the addition of fluorespheres at a known concentration it is possible to enumerate the number of CD34 cells you collect per graft. Most transplantations of hematopoitic material need this measure to set minimum dose requirements for transplantation.
CD4 CD8 Assay
The measure of ratios between Helper and Suppressor T cell populations is a means of tracking the disease progression in HIV to full blown AIDS. When a patient has been diagnosed with having the antibodies to HIV, this test becomes a routine application for tracking the progression of HIV. These tests are maintained for the life of that patient.
Monitoring Leukodepletion of Filtered Red Cell Products
In recent Guidelines handed down by the FDA, American Association of Blood Bankers and The Canadian Blood Services that have stated leukodepletion of red cell and platelet products should be monitored for quality assurance. Many blood bankers have been touting the utility of using cytometry as a means of measuring residual leukocytes in these products. Cytoquest Corp. in conjunction with the London Health Sciences Center and Beckman Coulter have developed a routine assay using microbead technology and DNA content for the assessment of leukodepleted products.
Sperm Chromatin Structure Assay For Fertility Assessment
Fertility Clinics typically assess semen quality by measuring sperm density, total count, motility and morphology. Clinics rarely measure sperm DNA integrity, perhaps because they are unaware of the availability of, or lack the instrumentation for, a rapid reliable and practical test. The sperm chromatin structure assay is one such test whose importance is becoming increasingly recognized. Fertility clinics in the greater Toronto area have expressed and interest in gaining adopting this test.
T Cell Crossmatch in Solid Organ Transplants
Prior to organ transplantation, the flow cytometric crossmatch is performed to detect antibodies that may be detrimental to the performance of the organ. The flow cytometric crossmatch assay involves incubating the donor’s lymphocytes with serum from the potential recipients of the graft.
Foeto-maternal bleeding can sensitise a Rhesus blood group D-ve mother to D+ve blood cells from the foetus. In a subsequent pregnancy, haemolytic disease of the new born child can be caused by the destruction of Rhesus D +ve blood cells of the foetus by maternal anti-D antibodies. Prophylactic anti-D given to the Rhesus D -ve mother shortly after delivery of a Rhesus D +ve child significantly reduces the incidence of anti-D sensitisation in the mother and has led to the virtual elimination of the disease from mothers so treated. Since the dose of anti-D given is related to the size of the foeto-maternal haemorrhage, quantitation of foetal-maternal haemorrhage is therefore important.
Quantitation is achieved by labelling the erythrocytes in a sample of maternal blood with FITC-conjugated, non-agglutinating anti-D antibodies (Nance et al., 1989). A population of as few as 0.1% foetal cells is sufficient to sensitise the parent so at least 500,000 cells should be analysed to obtain a statistically significant estimation.
Measurements on red blood cells (RBCs)
The measurements that can be made on red blood cells include:
• Detection and quantitation of RBC-bound proteins
• Quantitation of RBC-bound immunoglobulins
• Detection and quantitation of RBC antigens and antibodies
• Detection and quantitation of minor RBC populations, including the detection and quantitation of transfused RBCs and the detection and quantitation of fetal RBCs in maternal blood (see Section 7.8).
Platelet Counting and Function
Flow cytometry can be used to count platelets and also to measure their surface proteins (Harrison et al., 2001, 2005, Michelson, 2006). The latter change during activation of the platelets and can be used to measure their activation state. Platelets are easily activated and blood has to be taken and handled with care; for this reason, whole blood methods are generally preferred.
Platelet analysis by flow cytometry can have application in
• Identification of inherited disorders
• Monitoring of anti-platelet therapy
• Monitoring clinical course of disease
• Monitoring platelet production in thrombocytopenia
• Identification of patients at risk of thrombosis
• Accurate platelet counting in thrombocytopenia
• Diagnosis of heparin induced thrombocytopenia
Detection of Minimal Residual Disease
Minimal residual disease (MRD) was defined as disease beyond the limit of morphological detection using conventional microscopy. Patients with acute leukaemia were considered to be in remission when bone marrow samples contained <5% neoplastic cells. Flow cytometric methods can detect far lower levels of disease, which can be important in the clinical management of leukaemia. The residual tumour cells are detected using immunofluorescence of surface markers. A panel of at least three antibodies is used, the antibodies being selected on the basis of the immunophenotype of the original leukaemia.
Endothelial Cells and microparticles