Lymphoproliferative DIsorders and Automation 1 PDF

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Summary

Lymph nodes- This is the site of antigen recognition and processing. - It is also the site of lymphopoiesis and lymphatic drainage. - H&E stains are used on lymph tissues. - Reactive Lymphadenopathies, seen in reaction to infections and result in swollen lymph nodes.Chronic Lymphoproliferative D...

Description

MLSC-3121U, Clinical Hematology II Lymph nodes -

This is the site of antigen recognition and processing. It is also the site of lymphopoiesis and lymphatic drainage. H&E stains are used on lymph tissues. Reactive Lymphadenopathies, seen in reaction to infections and result in swollen lymph nodes.

Chronic Lymphoproliferative Disorders -

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Clonal proliferations of morphologically and immunophenotypically mature B or T lymphocytes. Cells come from one stem cell that is malignant and involves mature lymphocytes. Leukemia, disorders affecting the bone marrow and peripheral blood. Lymphomas, disorders affecting the lymph nodes or extramedullary sites. Can spill into the peripheral blood. Early Phase CLD, tumour cells are predominantly small with low proliferation rates. Transformation Phase CLD, frequent occurrence of extramedullary proliferation and an increase in large immature cells. Hodgkin’s Lymphoma, involves Reed-Sternberg cells (owl eyes) in lymph node imprints or cuts. A nodular lymphocyte-predominant Hodgkin’s lymphoma. Treatment depends on the stage of the disease but mainly involves chemotherapy and radiation. Non-Hodgkin’s Lymphoma, same as Hodgkin’s except there are no Reed-Sternberg cells. Staging is similar to Hodgkin’s lymphoma. Treated with radiation. Burkitt Lymphoma staging is different due to using bulk of tumour vs sites of involvement. Lymphocytic Lymphomas, Malignant population of lymphocytes arrested at a particular stage.

Chronic Lymphocytic Leukemia -

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Clonal population of morphologically and immunophenotypically mature B or T lymphocytes. Most common leukemia in adults and usually affects B cells. Peripheral blood and bone marrow lymphocytosis. Lymphocytes are everywhere and have big nuclei with less cytoplasm. Morphologically small or slightly larger than a normal lymphocyte. Bare nuclei seen as smudge cells are common. Albumin is added to an aliquot of blood to prevent smudge cells from occurring. An accumulating mass of lymphocytes, neutropenia, anemia, and thrombocytopenia are seen. This is because the overpopulated lymphocytes are taking over in resources and space. This can develop autoimmune disease leading to idiopathic thrombocytopenic purpura or autoimmune hemolytic anemia. B cells are abnormal and cannot produce normal IgG.

MLSC-3121U, Clinical Hematology II -

The lymphocytes are long lived, nonproliferating and immunologically dysfunctional. People usually live fairly long with this disease if monitored. Lab features include B cells lymphocytosis of 5.0 x 10^9/L, 30% lymphocytosis in the bone marrow, mature lymphocytes and normo/normo RBCs. There will also be a normal to low reticulocyte count, autoimmune hemolytic anemia and low platelets. Treatment is done to reduce signs and symptoms of disease with minimal discomfort or risk to the patient.

Burkitt’s Lymphoma -

Was once an acute leukemia (L3). Has a translocation of A14 chromosome that is common. Moderate amount of intensely basophilic cytoplasm, lipid vacuoles seen and proliferation rate very high with the volume doubling in one day. Highly aggressive and fatal if not treated. The disease is potentially and prognosis correlates with the bulk of the tumour.

Hairy Cell Leukemia -

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B lymphocytes with abundant cytoplasm, indented nuclei (bum cells) and fine hair-like cytoplasmic projections. Slight bone marrow fibrosis may be seen. TRAP Stain, The isoenzyme 5 in hairy cells are tartrate resistant and will react to the acid phosphatase stain. Acid phosphatase hydrolyzes a substrate. The substrate couples with a dye and precipitates at site of activity. Isoenzyme 5 is tartrate resistant, so when tartrate is added, all other enzymes except the hairy cell specific isoenzyme 5 will be inhibited. Hairy cells are resistant to tartaric acid and the substrate hydrolysis occurs and dye is precipitated at the site of activity. Activity when tartaric acid is added means a positive test for hairy-cells.

Mycosis Fungiodes and Sezary Syndrome -

Sezary syndrome is the leukemic phase of common T cell lymphoma mycosis fungiodes. Small to medium sized lymphoid cells with irregular nuclear outlines. The T cell nuclei in Sezary cell syndrome are called cerebriform (looks like a brain).

Multiple Myeloma -

Overproduction of abnormal plasma cells and involves the clonal population to produce identical mutated cells. Background staining on slide due to excess immunoglobulin is seen. Excess immunoglobulin also causes RBC rouleaux, decreased sedimentation rate and abnormal protein electrophoresis y spike. Plasma cells may be present in peripheral blood smear.

MLSC-3121U, Clinical Hematology II Flow Cytometry -

Monoclonal antibodies (mAbs) detect different antigens to confirm B or T lymphocytes. CD45 is on all hematology cells and is the hematology marker. CD34 is a good marker for immaturity since it is only on immature cells. T markers are the lower marker numbers (2,3,5,6) and B cells are the higher marker numbers (19,20,22).

AUTOMATION Hb Measurement -

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The hemoglobin molecule consists of 4 heme groups, 2 alpha and 2 non alpha chains. It is measured through its colour intensity in solution. Cyanmethemoglobin, the internationally recommended method that measures hemoglobin, methemoglobin, carboxyhemoglobin but not sulphemoglobin. It uses spectrometry at a wavelength of 540 nm. Hemoglobin in the ferrous state combines with cyanide to make methemoglobin. The potassium in potassium cyanide reacts with methemoglobin to produce cyanmethemoglobin which can be measured. The standard for this method is regarded as diluted whole blood. The original Hb the sample is obtained by multiplying the concentration stated on the standard’s label by the dilution factor applied to the sample. Measurement of Hb allows a direct comparison with the reference standard. All Hb can be measured except for sulphemoglobin. Sodium Lauryl sulphate, a method that replaces the cyanide containing Drabkin’s reagent to convert hemoglobin to hemoglobinsulphate.

Sources of Error -

Drabkin’s reagent is light sensitive, and light can affect the integrity of the reagent. A high WBC or lipemia that causes turbidity can interfere with the spectrophotometric method to cause false elevations. Hb S and C may cause the RBCs to not lyse and cause turbidity. Carboxyhemoglobin requires 1 hour to convert to cyanmethemoglobin.

Cell Counting -

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Wallace Coulter (1950), developed the electrical impedance used in modern day cell counters. The principle is based on the detection and measurement of changes in electrical resistance produced by cells as the travel through a small aperture. Cells are suspended in a conductive fluid like saline because cells do not conduct electricity. The fluid contains the cells is then pulled through an aperture. The aperture has electrodes that generate a current for the cells to break as they pass through. Breaks

MLSC-3121U, Clinical Hematology II

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are seen as spikes with sizes and quantities that are proportional to the size and quantities of cells. The pulses are separated into sizes and are associated with ells of proportional size for counting. Mercury Manometers, as mercury moves, based on vacuum pressure, a corresponding amount of fluid is pulled through the aperture.

Factors Affecting Electrical Impedance Cell Counting -

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Aperture Size, RBC/Plt aperture smaller than WBC aperture to increase Plt sensitivity. Protein Buildup, protein on the aperture can make it smaller and interfere with counting by decreasing cell flow and increasing impedance. Causes falsely low cell counts with falsely large cells. Carryover, cells can be carried over between samples, but it is minimized by internal cleaning. Coincidence Passing, two cells can pass through the aperture at the same time, but this can be corrected for mathematically. Recirculation, a backwash of cells can run back through the aperture, but this is prevented by a sweep-flow mechanism. Deformability, deformed RBCs can get stuck in the aperture and cause problems.

Mechanisms of Electrical Impedance Cell Counting -

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Hydraulics, includes the aspirating unit, dispensers, diluters, mixing chambers, baths, flow cells and a hemoglobinometer. Pneumatics, allow for vacuums and pressures to operate valves and moving the sample through the hydraulic system. Electrical Systems, includes the computer and digital equipment for sorting data and algorithmic corrections. Blood must be diluted for analysis. Two dilutions of a blood sample are made in saline. One dilution is delivered to the RBC bath for RBC and Plt count while the other is delivered to WBC bath for WBC count and Hb count. RBCs in the WBC bath are lysed with reagent to convert Hb into MetHb for measurement through spectrophotometry. Cells are sensed for 3 sequential count periods and the analyzer gathers the pulses generated for sorting. If insufficient data has been retrieved further accumulation takes place. Thresholds are used to determine the minimum and maximum size of cells that are counted.

MLSC-3121U, Clinical Hematology II -

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Hydrodynamic Focusing, the sample stream is surrounded by a sheath fluid as it passes through the central axis of the aperture. Creates laminar flow to focus the cell flow to allowing a single file flow of cells. Three Part differential, counts Lymphocytes, Mid cells (eosinophils, basophils and monocytes) and neutrophils.

RBC Histogram -

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RBCs are measured directly and the sample dilution contains RBCs, WBCs and Plt. However, it uses thresholds to determine which ones are RBCs. Plt are too small and not included in counting RBCs. WBCs are too small in quantity to interfere with RBC counts. The smallest population to the right of the RBC histogram main population represents the RBC doublets, triplets and WBCs. MCV, defined as the peak in the RBC histogram and the average size of a typical RBC. RDW, and indirect calculation of the average variability of size amongst the RBCs. It is derived from the histogram.

Platelet Histogram -

Platelets are also measured directly like RBCs are in the RBC histogram. The histogram forms a smooth curve from 2-20 fL and a fitted curve from 1-70 fL. The histogram has two main curves/humps and the count is usually derived from the fitted curve. MPV, the mean platelet volume.

WBC Histogram -

WBCs are directly measured to include all particles in the WBC bath that are larger than 35 fL. RBCs have already been removed using the lytic agent and platelets fall below the 35 fL threshold size and are ignored by the analyzer.

Hemoglobin Concentration -

Directly measured but calculated using a reagent blank and a mathematical equation. Freed hemoglobin is converted into a stable cyanide containing pigment measured at 525 nm. Absorbency of the sample is compared to a reagent blank....