Part III Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis AUTOMATED BLOOD CELL-COUNTING INSTRUMENTS O before automation - manual hemacytometer blood cell counts - spun hematocrits - spectrophotometrically determined hemoglobins - microscopic blood smear evaluations O using automated instrument - complete blood cell count (CBC) - platelet count - a five-part leukocyte differential - absolute reticulocyte count reticulocyte hemoglobin concentration (CHr or Ret He): -> assessment of iron incorporation into hemoglobin, functional availability of iron reticulocyte mean cell volume (MCVr) immature reticulocyte fraction (IRF) -> indicator of an erythropoietic response

Automation in Hematology and Hemostasis O two principles of blood cell counting 1. impedance (electrical resistance) - based on the increased resistance - the number of pulses indicate the blood cell count - amplitude (height) of each pulse: proportional to the volume (size) of the cell 2. optical light scattering - based on light scattering measurements - forward scatter: cell size - side scatter: cell complexity or granularity

Automation in Hematology and Hemostasis IMPEDANCE INSTRUMENTS Coulter Gen/S O by Beckman-Coulter O determine: CBC, five-part leukocyte differential, reticulocyte count O basic principle of operation - using EDTA anticoagulated blood sample, - four chamber 1. RBC/platelet dilution chamber - external electrode, three apertures, internal electrode (Fig. 36-1) - when blood cells pass through an aperture -> increase in resistance - threshold limit: for enumerating cells based on cell volume more than 36 fL -> erythrocyte 2-20 fL -> platelet the average of the counts from the three apertures

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis - RBC histogram (size distribution curve) (Fig. 36-2) visualization of changes in size within the erythrocyte population shift to the left: microcyte shift to the right: macrocyte - platelet histogram (Fig. 36-4) mean platelet volume (MPV), platelet distribution width (PDW) - obtainable data RBC count, MCV: by direct measurement RDW: derived from the RBC histogram (Fig. 36-3) Hct (MCV X RBC count): by calculated MCH (hemoglobin conc. / RBC count): by calculated MCHC (hemoglobin conc. / Hct): by calculated * hemoglobin concentration: obtained from the WBC/hemoglobin dilution chamber - platelet count: mean platelet volume(MPV), platelet distribution width(PDW)

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis 2. WBC/hemoglobin dilution chamber - determination of WBC count, hemoglobin concentration - WBC count: add lytic agent, directly measured lytic agent: lyse the erythrocytes, convert released hemoglobin to cyanmethemoglobin shrink the leukocyte cell membrane and cytoplasm - > 35fL -> leukocyte - three aperture - leukocyte histogram (Fig. 36-5) visualization of subpopulation of cells based on their relative sizes. lymphocytes: 35-90 fL monocytes: 90-160 fL granulocyte: 160-450 fL - system of region flags (Table 36-1) - hemoglobin concentration: absorbance of cyanmethemoglobin at 525 nm

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis 3. orbital mixing chamber - blood is mixed by gentle agitation with heated lysing reagent to remove the erythrocytes while leaving the leukocytes in their near native state - sent to the volume.conductivity.scatter (VCS) flow cell for determination of the five-part leukocyte differential volume: by impedance conductivity, scatter: evaluates internal physical and chemical constituents - contour gating (Fig. 36-6)

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis 4. heated reticulocyte dilution chamber - mixed with new methylene blue reagent - stained sample is mixed with an acidic, hypotonic solution elutes hemoglobin from the erythrocytes but permit precipitated RNA to remain spheres the erythrocytes (ligth scatter data -> more reproducible data) - sent to VCS flow cell classify reticulocytes vs mature erythrocytes - data absolute reticulocyte count, reticulocyte percentage, immature reticulocyte fraction reticulocyte mean cell volume (MCVr) O data compiling - corrects coincidence (two or more cells passing through the aperture at the same time) - Fig. 36-7 - software-generated flag or user-defined flag (definitive flag)

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis Coulter LH Series O provide a CBC, five-part leukocyte differential, reticulocyte count Wright-stained peripheral smear O utilize basic principle of the Gen.S instrument O additional parameter 1. nucleated red blood cell count (NRBC%), corrected WBC count (Fig. 36-8) - WBC scatterplot: detection of cells between the lymphocyte population and RBC ghosts - WBC histogram: the presence of cells to the left of the lymphocyte population - role out giant platelets or small lymphocytes - NRBC%: the number of nucleated red blood cells per 100 leukocytes - NRBC# = NRBC% X WBC count - NRBC: increase -> WBC count: falsely increase -> corrected WBC count

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis 2. RDW-SD - Fig. 36-9 - reflects the degree of anisocytosis - MCV does not affect the RDW-SD -> better indicator - reported in fL, reference interval: 39-47 fL - used in the differentiating iron deficiency anemia from heterozygous beta thalassemia - Fig. 36-10

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis Sysmex XE-2100 O impedence, radio frequency, absorption spectrophotometry, flow cytometry -> CBC, 5-part leukocyte differential, reticulocyte count O hemoglobin: measured using the sodium lauryl sulfate (SLS) method - lyse erythrocytes - convert ferrous iron to ferric iron (methemoglobin) by SLS - methemoglobin: combine with SLS -> SLS-hemichrome molecule - at 555 nm - advantage: decrease measurement time, reagent is cyanide-free O in the RBC/PLT channel: RBC, platelet count - using impedance with hydrodynamic focusing - MCV, MCH, MCHC, RDW-SD, RDW-CV, MPV

Automation in Hematology and Hemostasis O in the DIFF channel: leukocyte differential - hole in the cytoplasmic membrane of nucleated cells allowing a polymethine dye to enter the cell and bind to its DNA and RNA - forward scatter (cell size), side scatter (cell complexity) fluorescent intensity - Fig. 36-11 immature granulocyte (IG) count: metamyelocyte, myelocyte, promyelocyte O in the WBC/BASO channel - lyse erythrocytes - shear all leukocytes except basophils - Fig. 36-12 O in the IMI channel - mature leukocytes with phospholipid-rich membranes are completely lysed - immature myeloid cells (blasts) remain intact - provide the hematopoietic progenitor cell (HPC)

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis O in the NRBC channel - determine the presence of nucleated erythrocytes - NRBC and lymphocytes fall in the same cluster on the DIFF scattergram - remove the NRBC cytoplasmic membrane, shrink the nucleus - perforates only WBC cytoplasmic membrane, shape is not altered - fluorescent dye binds to nucleus and intracytoplasmic organelle - leukocyte: high fluorescent intensity and high forward scatter - Fig. 36-13 O in the RET channel - oxazine: binds to residual RNA within the reticulocytes - polymethine: binds to RNA and DNA within nucleated cells - differentiating mature erythrocytes, reticulocytes, platelets (Fig. 36-14) - immature platelet fraction (IPF): reticulated platelets increased size, higher fluorescence (Fig. 36-15) useful in diagnosis of thrombocytopenia associated with increased platelet destruction versus bone marrow failure - Fig. 36-16

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis Abbott CELL-DYN Sapphire O for CBC, a five-part leukocyte differential, reticulocyte O flow cytometry, fluorescence staining, impedance O in the hemoglobin dilution cup - lyse erythrocytes - convert hemoglobin to a single chromogen by forming a complex with imidazole cyanide-free reaction - determined spectrophotometrically at 540 nm

Automation in Hematology and Hemostasis O in the WBC dilution cup - total WBC count, nucleated RBC, five-part leukocyte differential - flow cytometry (light scattering, fluorescence (for NRBC) - using multi-angle polarized scatter separation (MAPSS) (Fig. 36-17) 0 degree light scatter (forward scatter): cell size 7 degree light scatter: cell complexity 90 degree light scatter (side scatter): nuclear lobularity 90 degree depolarized light scatter: cytoplasmic granularity - 0 degree (size) versus 7 degree (complexity): differentiating of neutrophil, monocyte, lymphocyte (Fig. 36-18) 90 degree D (granularity) vs 90 degree (lobularity) differentiating of eosinophil, neutrophil - NRBC is clearly distinguished from leukocytes strip the cytoplasmic membrane from nucleated erythrocytes

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis O in the RBC/PLT dilution cup - erythrocyte/platelet dilution sphere the erythrocytes, RBC, platelet count by impedance, light scattering - reticulocyte dilution fluorescence (RNA), 7 degree light scatter (cell complexity) (Fig. 36-19) O Fig. 36-20 O fluorescence immunophenotyping method O immuno T-cell assay - using anti-CD3 (T cell), CD4 (T helper cell), CD8 (T cytotoxic cell) - scatter plot of 0 degree vs 7 degree O immuno PLT (CD61) assay - using anti-CD61 - when platelet count is very low, leukocyte or erythrocyte fragments interfere

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis LIGHT-SCATTERING INSTRUMENTS Siemens Healthcare ADVIA 120 O CBC, five-part leukocyte differential, reticulocyte count O five measurement channel (1) the erythrocyte/platelet channel - RBC, platelet count - isovolumetric sphering (sphere and fix the blood cells without altering their volume) of the erythrocytes and platelets -> eliminate cell volume errors due to variation in erythrocyte shape - counted and sized by high-angle (5-15): hemoglobin concentration low-angle (2-3): cell volume (size) (2) the hemoglobin channel - lyse erythrocyte - converted to cyanmethemoglobin

Automation in Hematology and Hemostasis (3) the peroxidase channel - identify neutrophil, monocyte, eosinophil by the degree of peroxidase positivity and forward light scatter - lymphocyte and large unstained cell (LUC): forward light scatter - neutrophil: upper right quadrant eosinophil: lower right quadrant monocyte: the cental triangle region lymphocyte: adjacent to the y-axis in the center left quadrant LUC: upper left quadrant (4) the basophil/lobularity channel - lyse erythrocyte and platelet - strips all leukocytes except basophils of their cytoplasm - determined by high and low-angle scatter - each population is identified by its position, area, and density - normal: worm type mononuclear cell: head, neutrophil: tail, basophil: large, above the worm

Automation in Hematology and Hemostasis (5) the reticulocyte channel - stained with oxazine 750 - light scattering: high-angle light -> hemoglobin concentration low-angle light -> size - absorbance: RNA content O no reporting NRBC count O Fig. 36-21 Siemens Medical ADVIA 2120 O hemoglobin determination: cyanide-free methodology

Automation in Hematology and Hemostasis

Automation in Hematology and Hemostasis AUTOMATED COAGULATION INSTRUMENTS O based on electromechanical or optical density methods of clot detection O plus chromogenic and immunologic methods ELECTROMECHANICAL INSTRUMENTS BBL FibroSystem O detect completion of an electrical circuit between two electrodes when a clot forms O fibrometer probe - clot detector - two sensory electrodes (stationary and moving) - fibrin clot formation -> creation of electric circuit: from stationary electrode through the reaction mixture to fibrin clot to moving electrode

Automation in Hematology and Hemostasis Diagnostica Stago STArt 4 Clot Detection Instrument O based on the increasing viscosity of plasma as clot formation occurs O detect decrease in movement of an iron ball in an electromagnetic field when a clot forms - increasing viscosity is detected by the movement of an iron ball located at the bottom of the reaction cuvette - increase viscosity -> decrease in ball movement OPTICAL DENSITY INSTRUMENTS Trinity Biotech Coag-A-Mate XM O detect sudden increase in optical density(absorbance) occurring with fibrin formation

Automation in Hematology and Hemostasis CHROMOGENIC/CLOT DETECTION INSTRUMENTS O combine clot-based detection principle with chromogenic analysis and immunological techniques O the enzyme of interest cleaves the chromogenic substrate at a specific site, releasing the chromophore tag Trinity Biotech AMAX Destiny Plus O electromechanical, optical density, chromogenic, and immunologic O electromechanical measurement of clot formation - as the fibrin clot forms, fibrin strands draw the ball away from its steady-state position - accurate results are obtained on icteric samples, lipemic samples, and samples from patients on medication that affect optical measurement

Automation in Hematology and Hemostasis O optical density - monitor the change in absorbance over time at 405 nm - the effects of icteria and lipemia on the clotting time is minimized O chromogenic assay - paranitroaniline (chromophore tag) at 405 nm O immunologic assay - detect and quantitate the presence of certain coagulation proteins or products - D-dimer assay detect the increase in absorbance that occurs as immune complex form between the sample's D-dimers and the monoclonal antibodies, thus aggregating the microparticles

Automation in Hematology and Hemostasis Diagnostica Stago STA Compact O electromechanical principle, chromogenic and immunological assay O electromechanical clotting assay - as the fibrin clot forms, the increasing viscosity of the reaction mixture -> decrease the ball's oscillation - clotting time are not affected by lipemic or icteric samples because the determination does not depend on light absorbance O chromogenic assay - chromogen: paranitroaniline O immunologic assay - measure the concentration of von Willebrand factor - microlatex beads coated with rabbit anti human vWF to aggregate or clump -> increasing the absorbance