Flow Cytometry and Quality Assurance PDF

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Summary

Flow Cytometry- A technique used to count, sort and examine microscopic particles suspended in a stream of fluid. - This measures the property of cells using a photomultiplier measuring device. The Beckman Coulter analyzer is the most common clinically used flow cytometer, while the BD is used more ...

Description

MLSC-3121U, Clinical Hematology II Flow Cytometry -

A technique used to count, sort and examine microscopic particles suspended in a stream of fluid. This measures the property of cells using a photomultiplier measuring device. The Beckman Coulter analyzer is the most common clinically used flow cytometer, while the BD is used more for research.

Samples -

Most of the time, bone marrow aspirates and peripheral blood is used for flow cytometry. However, sometime sample may be soft tissue (lymph nodes, liver) which are ground into a mix of cells using a sieve. CSF may also be analyzed in flow cytometry.

CD Nomenclature -

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A system that is used to categorize monoclonal antibodies known as CDs. Each CD antibody will have its own specific CD cell antigen that it binds with. The CD is a designation of the monoclonal antibody to which antigen it binds. Granulocytes will have a mix of CD antigens that are unique to them, allowing us to differentiate them. Serves to standardize the use and naming of laboratory antibodies to cell surface molecules. CD45 is on all myelocytes and lymphocytes.

Sample Processing -

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Ficoll Method, uses diluted whole blood and a density gradient fluid to separate WBCs from RBCs based on density. Whole Blood Lysis, uses whole blood or diluted bone marrow that is stained with monoclonal Abs. RBCs are lysed with a lysing agent and the sample is washed, resuspended and run. Allows for a presentation of all the cells in the specimen and prevents loss of the population of interest/antigenic expression. More commonly used. For flow cytometry, RBCs must be lysed removed to isolate the WBCs. Once isolated, the monoclonal florescent Abs will bind to the cell based on their CD designation. This allows us to differentiate cells based on the colour of fluorescence.

Single Platform Testing -

Sample undergoes whole blood lysis and uses a calibrated bead of known concentration. This allows for the qualitative values of cell populations to be reported.

MLSC-3121U, Clinical Hematology II

Hydrodynamic Focusing -

Uses an isotonic sheath solution that runs around the sample tube to meet it at the tubes aperture. The cell suspension is then introduced into the sheath fluid flow which guides the cells into the laser one at a time.

Fluorescence Detection -

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The labelled Abs are struck with a single wavelength, which will be emitted back at a different wavelength based on the dye. Compensation, some of the emitted wavelengths overlap each other which causes some difficulty when identifying cells. Compensation serves to remove the amount of fluorescent in each detector that does not originate from a signal produced by that specific fluorochrome. This is done in the computer when it calculates what fluorescence overlaps where and compensates for it. Compensation verification looks for positivity and negativity to the monoclonal Ab used to stain the WBCs. Samples with known amounts of labelled WBCs is used to make sure the analyzer can properly compensate for the florescence bleed-over. Photomultipliers, display data in a visual and statistical manner. Uses forward scatter to show size and side scatter to show complexity through fluorescent detection.

CD45 Gating -

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Used to obtain one cell population of interest (usually mononuclear cells) to gain information on them. Helps to define cell lines based on maturity by purifying the cell population for gated analysis. CD45 is found on all hematology cells and used to determine the population of interest. Gating can be done by graphing the populations based on forward scatter, side scatter and/or CD45. The analyzer will always measure all cells in the sample so you can go back and re-gate to isolate different cell populations, but it will only display what is not gated out.

Flow Cytometry and Acute Leukemia -

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In acute leukemia, there is a rapid increase in immature blood cells. We do not always know which blasts are proliferating and being affected. Immediate treatment is needed for these patients since the accumulation and proliferation of malignant cells is very fast. Can result in them spilling over into the blood stream and metastasizing in the different organs. Flow cytometry also us to define the type of blasts present. This is done through the use of myeloid, monocytic, and lymphoid panels. Each panel has different CD antigens, with the exception oof CD45 being present in all of the hematology cells.

MLSC-3121U, Clinical Hematology II -

There are five monoclonal antibodies used for each panel each producing a different colour. Lymphoproliferative Disorders, can be very hard to differentiate the T and B lymphoproliferation. Flow cytometry confirms whether it is B or T cell related. The CD20 antigen is found on B lymphocytes. There are also separate panels specifically made for detecting lymphoproliferative disorders (CLL, MCL, HCL, etc.).

PCR -

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Amplifies a single target gene by replicating it to a detectable level. Reverse Transcription PCR, requires RNA for analysis. cDNA acts as a template for replication. Used to analyze the BCR-ABL gene in CML. Real-Time PCR, uses similar techniques as RT-PCR but involved tagging a fluorescent label onto the DNA to avoid extra visualizations with electrophoresis. After replicated DNA is obtained from PCR it can be visualized on gel electrophoresis, restriction endonucleases, nucleic acid hybridization, DNA sequencing and fluorescent probes. Restriction Fragment Length Polymorphism, the use of restriction endonuclease to identify gene mutations. It detects the changes in the length of resulting bands after they are restricted. Used to identify actor V Leiden. Nucleic Acid Hybridization, uses Southern Blot analysis (electrophoresis). Creates smears of DNA on the electrophoresis. More common way of looking at DNA. o Northern Blot, RNA is electrophoresed and probed. Used to study gene expression and is not diagnostic. DNA Sequencing, reads the entire gene sequence of nucleic acids.

Quality Assurance -

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Includes training personnel, using the right reagents and equipment, and QC itself. Analyzers are a large part of quality assurance as they require different control levels to be run usually after major maintenance and less during lot changes. Pre-Analytical Variables, things that affect the sample prior to testing. o Proper Patient ID o Tube Labelling o Sample Mixing o Anticoagulants o Sample Collection from IV o Medications o Delievry Time o Previous Blood Transfusion. Post-Analytical Variables, things that verify the validity of the sample after testing. o Delta Checks

MLSC-3121U, Clinical Hematology II

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o Relasing Results o Calling Critical Results o Reflex Testing o Specimen Re-Checking for Clots Critical Results, include WBC, Hb and Plts. These tests have critical since they have levels that can cause detrimental effects to the patient if they go uncalled. Bull’s Algorithm, in a batch of 20 plotted samples the averages out the highs and lows to form a constant (x-bar B). The constant can be compared to a range and target values. Used to troubleshoot instruments. If MCV is out of range, the RBC/Plt bath has a problem. If MCH is out of range, the Hgb or the RBC bath has a problem. If the MCHC is out of range, it could be either bath or both baths. Out of range x-Bar B can also be caused by the patient population (ex. high thalassemia population cause a lot of micro hypo in the batch of 20). Delta Checks, when a patient’s current result differs from the immediate previous result, it can be caused by several things. o Clerical Error o Analytical Error o Specimen Mix-Up o Change in Patient Physiological State When a delta check occurs, the sample is often reflex tested since it can indicate something that is wrong with the patient (ex., low Hgb may be hemoglobinopathy or IDA. Can be reflexed for SPE, Ferritin and/or manual differential). Ideal Hematology Controls, should be: o Inexpensive o Stable for a Long Time o Easily Suspended o Non-Agglutinating o Similar to Blood in Proportions and Cell Size o Assayed Independently Rule of Three, HgB x 3 = Hct +/- 0.03. RBC x 3 = Hgb. MCHC between 320 and 360. High HgB that fails the rule of three may be caused by lipemia or icteric (absorbance positive interference). Low Hct that fails the rule of 3 is caused by RBC agglutination. Warm the sample up to break up Ag-Ab from cold agglutinating Abs....