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![Automation in Haematology
• Automation provides both greater accuracy and greater precision than
manual method.
• Over the last 20 years, instrumentation has virtually replaced manual cell
counting .
• Haematology analyzers typically provide the eight standard haematology
parameters (Full blood count [FBC] plus a three part or five part differential
leucocyte counts in less than one minute on 100µl of whole blood.
• Automation thus allows for more efficient workload management and more
timely diagnosis and treatment of disease.](https://image.slidesharecdn.com/automationinhaematology-haemiiimlh305-250710065548-d74da953/75/Automation-in-Haematology-HAEM-III-MLH-305-pptx-2-2048.jpg)
![Optical Scatter
• Light scatter results form the interaction between the processes of
absorption, (diffraction bending around corners or surface of cell), refraction
(bending because of a change in speed), and reflection (backward rays caused
by obstruction).
• The detection and conversion of scattered rays into electrical signals is
accomplished by photo detectors (photodiodes and photo multiplier tubes
[PMTs] at specific angles.](https://image.slidesharecdn.com/automationinhaematology-haemiiimlh305-250710065548-d74da953/75/Automation-in-Haematology-HAEM-III-MLH-305-pptx-13-2048.jpg)
Automation in Haematology - HAEM III MLH 305.pptx
- 1. Automation in Haematology MR. PRINCEALLOTEY ATU HAEM III MLH 305 1ST DECEMBER, 2024
- 2. Automation in Haematology •Automation provides both greater accuracy and greater precision than manual method. • Over the last 20 years, instrumentation has virtually replaced manual cell counting . • Haematology analyzers typically provide the eight standard haematology parameters (Full blood count [FBC] plus a three part or five part differential leucocyte counts in less than one minute on 100µl of whole blood. • Automation thus allows for more efficient workload management and more timely diagnosis and treatment of disease.
- 3. Automation in Hematology •Automated haematology analyzers can rapidly analyze whole blood specimens for the full blood count (FBC). • Results include red blood cell (RBC) count, white blood cell (WBC) count, platelet count, hemoglobin concentration, hematocrit, RBC indices, and a white cell differential. • The best source of information about the various instruments available is the manufacturers’ product information literature.
- 4. General principles ofhaematology instrumentation • Despite the number of haematology analyzers available form different manufacturers and with varying levels of sophistication and complexity, two basic principles of operation are primarily used: electronic impedance (resistance) and optical scatter. • Electronic impedance, or low-voltage direct current (DC) resistance, was developed by Wallace Coulter in the 1950s and is the most common methodology used. • Radio frequency (RF), or high-voltage electromagnetic current resistance, is sometimes used in conjunction with DC electronic impedance.
- 5. General principles ofhaematology instrumentation • Technicon Instruments introduced dark field optical scanning in the 1960s, and Ortho Diagnostics systems followed with a laser-based optical instrument in the 1970s. • Optical scatter, utilizing both laser and non-laser light, is frequently used on today’s haematology instrumentation
- 6. Electrical Impedance • Theimpedance principle of cell counting is based on the detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture. • Cells suspended in an eclectically conductive diluent such as saline are pulled through an aperture (orifice) in a glass tube. • In the counting chamber, or transducer assembly, low-frequency electrical current is applied between an external electrode (suspended in the cell dilution) and an internal electrode (housed inside the aperture tube).
- 7. Electrical Impedance • Electricalresistance between the two electrodes, or impedance in the current, occurs as the cells pass through the sensing aperture, causing voltage pulses that are measurable. • Oscilloscope screens on some instruments display the pulses that are generated by the cells as they interrupt the current. • The number of pulses is proportional to the number of cells counted
- 9. Electrical Impedance • Thesize of the voltage pulse is directly proportional to the size (volume) of the cell, thus allowing discrimination and counting of specific-sized cells through the use of threshold circuits. • Pulses are collected and sorted (channelized) according to their amplitude by pulse height analyzers. • The data are plotted on a frequency distribution graph, or size distribution histogram, with relative number on the y-axis and size (channel number equivalent to specific size) on the x-axis.
- 10. Electrical Impedance • Thehistogram produced depicts the volume distribution of the cells counted. • Size thresholds separate the cell populations on the histogram, with the count being the cells enumerated between the lower and upper set thresholds for each population. • Size distribution histograms may be used for the evaluation of one cell population or subgroups within a population. • The use of proprietary lytic reagents to control shrinkage and lysis of specific cell types, allows for separation and quantitation of white blood cells into three populations (lymphocytes, mononuclear cells, and granulocytes) for the “three-part differential” on one size distribution histogram.
- 11. Optical Scatter • Opticalscatter may be used as the primary methodology or in combination with other methods. • In optical scatter systems (flow cytometers), a hydro-dynamically focused sample stream is directed through a quartz flow cell past a focused light source. • The light source is generally a tungsten-halogen lamp or a helium-neon laser (Light Amplification by Stimulated Emission of Radiation). • Laser light, termed monochromatic light since it is emitted as a single wavelength, differs from bright field light in its intensity, its coherence (i.e. it travels in phase), and its low divergence or spread.
- 12. Optical Scatter • Thesecharacteristics allow for the detection interference in the laser beam and enable enumeration and differentiation of cell types. • Optical scatter may be used to study RBCs, WBCs, and platelets. • As the cells pass through the sensing zone and interrupt the beam, light is scattered in all directions.
- 13. Optical Scatter • Lightscatter results form the interaction between the processes of absorption, (diffraction bending around corners or surface of cell), refraction (bending because of a change in speed), and reflection (backward rays caused by obstruction). • The detection and conversion of scattered rays into electrical signals is accomplished by photo detectors (photodiodes and photo multiplier tubes [PMTs] at specific angles.
- 14. Optical Scatter • Lensesfitted with blocker bars to prevent non-scattered light from entering the detector are used to collect the scattered light. • A series of filters and mirrors separate the varying wavelengths and present them to the photo detectors. • Photodiodes convert light photons to electronic signals proportional in magnitude to the amount of light collected. • Photo multiplier tubes are used to collect the weaker signals produced at a 90 degree angle and multiply the photoelectrons into stronger, useful signals.
- 15. Optical Scatter • Analog-todigital converters change the electronic pulses to digital signals for computer analysis • Forward-angle light scatter (0 degrees) correlates with cell volume or size, primarily because of diffraction of light. • Orthogonal light scatter (90 degrees), or side scatter, results from refraction and reflection of light from larger structures inside the cell and correlates with degree of internal complexity.
- 16. Optical scatter • Forwardlow-angle scatter (2-3 degrees) and forward high-angle scatter (5-15 degrees) also correlate with cell volume and refractive index or with internal complexity, respectively. • Differential scatter is the combination of this low- and high-angle forward light scatter, primarily utilized on Bayer systems for cellular analysis. • The angles of light scatter measure by the different flow cytometers are manufacturer and method specific.
- 18. Light Scatter • Sidelight scatter: light scattered at a 90◦ angle from the particle defines internal complexity and granularity of the particle. • Neutrophils and eosinophils produce a great deal of side scatter due to their cytoplasmic granules. • Forward scatter: light that continues in the forward direction relates to the particle size. • Large cells such as monocytes and neutrophils produce more forward scatter than nRBCs, and normal lymphocytes.
- 19. Light Scatter Plot SideScatter
- 21. Parameters • Red bloodcell values provided by blood cell counters : • Haemoglobin (Hb) • Haematocrit (Hct) or packed cell volume (PCV) • MCV • MCH • MCHC • Red cell distribution width (RDW) • RBC count • White cell differential count • Platelet measurements: • Platelet count • Mean platelet volume (MPV) • Platelet distribution width (PDW) • Plateletcrit
- 22. Red Cell Indices •The MCV, MCH, and MCHC are referred to as red cell indices. • They are calculated from the results of red cell count, Hb concentration and PCV. • Red cell indices are widely used in the classification of anaemia. • Measurements directly obtained by most blood cell counters are RBC count, Hb, MCV and RDW. • PCV, MCH and MCHC are calculated from combinations of the primary measurements. • MCV is obtained either from the mean height or a selected span of the pulses generated during the red cell count or from the sum of the pulse height divided by the number of pulse generated during the count.
- 23. Red Cell Indices •Hb is measured as cyanmethaemoglobin (HiCN) in a standard procedure. • PCV is deduced from the red cell count and MCV • MCH is deduced from the Hb and red cell count. • MCHC is calculated from the measured Hb and the deduced PCV.
- 24. Classification of Anaemia •Anaemia can be classified based on red cell indices as microcytic or macrocytic anaemia. • MCV describes the size of the RBC (86 ± 10 fL). Values above 100 fl indicates macrocytosis, values below 70 fL indicates microcytosis. • MCV is rarely higher than 150 fL or lower than 50 fL. • MCV values are often raised in megaloblastic anaemia(commonly due to folate or vitamin B12 deficiency.)
- 25. Classification of Anaemia •MCH defines the haemoglobin content of RBC (29.5 ± 2.5 pg). • Hypochromia results from values which are usually below 27 pg. • MCH is rarely higher than 50 pg nor lower than 15 pg. • Low MCH occur in iron deficiency anaemia and the thalassemias.
- 26. Classification of Anaemia •MCHC is often not used in the classification of anaemia because: • It may be normal or diminished in macrocytic • It is often diminished in microcytic hypochromic anaemias.
- 27. Classification of Anaemia •RDW is a measure of the degree of anisocytosis, the extent of the red cell size variation distribution. • Its unit of measurement is the coefficient of variation (CV) or standard deviation (SD). • Unit of CV is % whilst unit of SD is fL(femtolitre) • The normal range as CV is 12.8 ± 1.2%. Values above 15% are regarded as increased. • The normal range as SD is 45 ± 8 fl.
- 28. Classification of Anaemia •It is useful in distinguishing between iron deficiency and thalassaemia trait. • A low MCV with a normal RDW suggest thalassaemia trait. • A low MCV with an increased RDW indicates iron deficiency • RDW is also useful in differentiating high MCV due to aplastic anaemia from that due to megaloblastic anaemia. • RDW is normal in aplastic anaemia but high in megaloblastic anaemia
- 29. White Cell Count •The WBC parameters provided by cell counters are total number of leucocytes and the differential count. • Normal range for WBC depends on age and other variables. • Counts are performed on diluted blood in which red cells are either lysed or rendered transparent • Differential count provided by some blood cell counters is calculated based on size and the granularity of the WBC. • .Differential counting could be either a three-part or five to seven-part differential count.
- 30. White Cell Count •3-part differential count assign cells to categories usually designated: • 1. Granulocytes or large cells which include eosinophils and basophils • 2. Lymphocytes or small cells • 3. Monocytes (mononuclear cells or middle cells) • 5 to 7 –part differential count classifies cells as neutrophils, eosinophils, basophils, lymphocytes and monocytes. • Automated instruments performing 5 to 7-part differential count are able to ‘flag’ or reject counts from samples with nucleated RBCs, myelocytes, promyelocytes, blasts and atypical lymphocytes.
- 31. Platelet Measurements • Plateletmeasurements provided by blood cell counters are: • Total number of platelets • Mean platelet volume (MPV) • Platelet distribution width • Plateletcrit
- 32. Platelet Measurements Platelet Count:the reference value 150 – 450 x 109 /l Mean Platelet Volume: reference range 9 – 13 fl. This depends on many variables, one of which is platelet count. • The higher the platelet count, the lower the normal value of the MPV. • Low platelet counts with normal MPV occur in immune thrombocytopaenic purpura (ITP). • Thrombocytopaenia with high MPV values occur in Bernard-Soulier syndrome (characterized with giant platelets).
- 33. Platelet Measurements • Myeloproliferativedisorders are associated with both high platelet count and MPV. E.g Essential thrombocythaemia. CML Platelet Distribution Width (PDW): this is a measure of platelet anisocytosis. • PDW has been found to be useful in distinguishing between essential thrombocythaemia in which the PDW is increased from reactive thrombocytosis in which the PDW is normal. Plateletcrit : this is the product of the MPV and the platelet count. • It is indicative of the volume of circulating platelet in a unit volume of blood.
- 34. Sources of Errorin cell count • Cold agglutinins • Low red cell counts and high MCVs can be caused by an increased number of large red cells or red cell agglutinates. • If agglutinated red cells are present, the automated hematocrits and MCHCs are also incorrect. • Cold agglutinins cause agglutination of the red cells as the blood cools.
- 35. Sources of Errorin Cell Count • Cold agglutinins can be present in a number of disease states, including infectious mononucleosis and mycoplasma pneumonia infections. • If red cell agglutinates are seen on the peripheral smear, warm the sample in a 37°c heating block and mix and test the sample while it is warm.
- 36. Sources of Errorin Cell Count Fragmented Cells or very microcytic red cells 1. These may cause red cell counts to be decreased and may flag the platelet count as the red cells become closer in size to the platelets and cause an abnormal platelet histogram. 2. The population is visible at the left side of the red cell histogram and the right end of the platelet histogram
- 37. Sources of Errorin Cell Count • Platelet clumps and platelet satellitism • These cause falsely decreased platelet counts. • Platelet clumps can be seen on the right side of the platelet histogram. • Decreased platelet counts are confirmed by reviewing the peripheral smear. • Always scan the edge of the smear when checking low platelet counts.
- 40. Sources of Errorin Cell Count • Giant Platelets These are platelets that are close to or exceed the size of the red cells. They cause the right hand tail of the histogram to remain elevated and may be seen at the left of the red cell histogram.
- 43. Sources of Error •Nucleated RBCs These interfere with the WBC on some instruments by being counted as white cells/lymphocytes
- 44. Sources of Error •Measuring Hemoglobin • Anything that will cause turbidity and interfere with a spectrophotometry method. • Examples are a very high WBC or platelet count, lipemia and hemoglobin types that are resistant to lysis, such as hemoglobin S and C.
- 45. Summary • Automated techniqueshave replaced manual techniques in determining the various elements of the Full blood count. • This is so because they are not as laborious as the manual techniques and many samples can be analyzed within a short time. • A drawback is the difficulty on recognizing the existence of erroneous results when measurements are carried out on a single machine, as the error may be constant in the particular instrument.
- 46. Summary • Counts mayvary from instrument to instrument and even with different models of the same instrument • It is necessary to calibrate the instrument from time to time and to employ quality assurance procedures to check on precision and accuracy. • It is important to prepare a blood film for every sample analyzed by the electronic counter since examaination of such films will assist in identifying certain errors.
- 47. Other Automated Techniques •Other automated techniques introduced in haematology include: • Automated system for staining of blood smears. • For coagulation studies several automated and semi-automated systems are available. Prothrombin time and activated partial thromboplastin time determinations can be done automatically on various instruments. • Serology – ABO and Rh blood grouping and crossmatching
- 48.
Editor's Notes
- #8 Coulter principle of cell counting based on Electronic impedance.
- #32 When the platelet count is higher, that the platelet sizes are smaller, making the MPV lower normal. Low platelet counts with normal MPV in ITP – platelets have been produced normally but being destroyed in circulation due to idiopathic causes.
- #37 Platelet satellitism- platelet revolving around neutrophils just like the planets revolving around the sun
- #38 Normal RBC size 76 – 90 fL.
- #42 Schistocytes are fragmented red cells, typically irregularly shaped, jagged and have two pointed ends.