“ MP “, multiple peaks Note: Parameter: PLT, MPV, PDW and P-LCR is flagged. platelet anisocytosis PLt count normal Re-collect sample with PLT aggregation PLT count false low sodium citrate
WBC Histogram WBC channel –(WBC and PLT) Curve start and end at the basis line LD: Variable < 30 fL PLT /LD/ WBC
WBC HISTOGRAM ❑ Neutrophilia ❑ Lymphocytosis ❑ Monocytosis ❑ Eosinophilia ❑ Basophilia AB CD
WBC channel –(WBC and PLT) “ WL “, Curve does not begin “ WU “, Curve does not end at at the basis line the base line • PLT Clumps EDTA- Incompatibility coagulated Sample High Leukocyte count Leukocyte Agglutination • High osmotic resistant (Erythrocytes not lysed) • Erythroblasts • Cold agglutinate
No T1 -High no. of large or atypical lymphocytes No T2 Neutrophilic hypolobation eg. Pelger Huet and pseudo Pelger Huet anomaly.
✓ WBC count unusually high Must do: ✓ Smear found NRC Corrected WBC count = (measured WBC Count x 100) / (100 + NRBC count ) NRBC Count: The number of NRBC per 100 leukocytes.
Resolved -Incubate 37 oC - Wash with NSS Leukocyte Agglutination
Errors in Hematology analyzer
ERRORS 1. Recirculation error 2. Coincidence error Cells that pass through the aperture simultaneously, 3. Non central flow error Cell pass through the aperture off centre produce aberrant impulses and appear larger than their actual size
Hydrodynamic focusing and Laminar flow
Cold agglutinins Patient PB smear With cold เม็ดเลือดแดงเกาะตัวทีอ่ ณุ หภูมหิ ้อง หรือ agglutinin Normal เยน็ เนื่องจากมี antibody ตอ่ เมด็ เลือดแดง ชนดิ IgM ERROR in CBC RBC count Low MCV High Bad RBC histogram Good indication : not relate to HCT from microhematocrit method : สามารถมองเห็นการเกาะตวั ของเมด็ เลือดแดงท่ีหลอด EDTA (A)
WORMED CITRATE EDTA Ratio 1.1 207,900/μL ครั้งแรก EDTA- Induced Thrombocy topenia
pre-analytic errors Cause Pseudo thrombocytopenia ✓ over-filled or under-filled check the tube (PTCP) EDTA tubes) do a delta check a smear review ✓ clotted specimens ✓ a time delay between sample collection and testing in vitro agglutination of platelets ✓ cold agglutinins, multiple myeloma ✓ Infections ✓ anticardiolipin antibodies ✓ high immunoglobulin levels ✓ abciximab therapy and EDTA induced pseudothrombocytopenia. (EDTA-PTCP)
OTHER ERROR IN DOCUMENT
Must to do before running the sample CBC specimens must be checked for clots (visually, by applicator sticks, or by automated analyzer histogram inspection or flags), significant in-vitro hemolysis and interfering lipemia before reporting results. • CBC processing, either automated or manual, should be done 8 hours -24 hours of sample collection, Blood samples must be adequately mixed before analysis.
Quality Control Quality Assessment: Adequate control of the pre & post analytical from sample collection to report dispatch. Quality Control: Measures that must be included during each assay run to verify the test working properly. Proficiency Testing: Determines the quality of results generated by lab. Terminologies
Delta Checks a quality control tool that involves the comparison of laboratory test results with results obtained on previous samples from the same patient Low day-to-day variation red blood cell (RBC) indices electrolytes liver function tests.
Internal Quality control: Continuous evaluation of the reliability of the daily works of the lab with validation of tests. External Quality Control: Evaluation by an outside agency of between- laboratory & between-method comparability. TYPES
CBC Quality Control Commercial Controls: • 3 levels (low, normal, high) • Values stored in instrument computer • Levey-Jennings graph generated and stored for each parameter • Delta Checks –Current values compared to most previous result – Differences greater than the limits set within the LIS are flagged
Controls & Calibrators Controls: Substances used to check the precision . o Analyzed either daily or along each batch. o same test properties as blood samples. o Stabilized anticoagulated whole blood or pooled red cells. o 3 conc.-high, normal, low o Levey-Jennings graph generated and stored for each parameter Calibrators: Check the accuracy. Value assigned to them by a reliable ref. center.
Control Values and Decision ▪ using Westgard Control Rules ▪ Use premise that 95.5% of control values should fall within ±2SD ▪ Commonly applied when two levels of control are used Levey-Jennings Chart
What to do when Control Value is out of limit? precision of routine work can be monitored by performing duplicate tests on patient samples SD of differences between results on 20 duplicate samples is determined and +2SD limits specified Subsequent duplicate values should be within these defined limits. A Patient data can also be used to monitor precision in a laboratory performing >100 samples a day The use of stable controls (method of chice) Day-to-Day variation in MCV, MCH and MCHC should be analyzed using Bull's algorithm -the software of many auto analyzers.
• An instrument problem is differentiated from a specimen- related problem by running a control. • If the control results are acceptable, the problem is probably specimen-related. • Check for: – clots – hemolysis – lipemia
OUT OF CONTROL!!! Repeat the assay ( One time occurrence ) –Check for trends (Delta check) (from Levy Jennings chart) –Check integrity of material –Troubleshoot –Verify instrumentation
Instrument Problems If the control shows similar problems, it indicates an instrument problem. – Electronic? – Pneumatic / Hydraulic? – Reagent? A reagent problem precipitate in the reagent tubing. expiration dates of the reagents in use
Calibration 1. to compensate for any inaccuracies of the pneumatic hydraulic and electric systems 2. Calibration fine tunes your hematology analyzer and provides the most accurate results possible. 3. Automated Hematology analyzers should be calibrated using calibrators„ that have traceability to standard reference material or methods. 4. Controls are not used for calibration Calibration • Never adjust to a specific value for an individual sample. • For best performance, calibrate all the CBC parameters. [The WBC differential is calibrated at the factory. They do not require calibration in the laboratory] Blood Sample for Calibration • 4 ml specimen are obtained from three hematological normal volunteer in k2EDTA Hb estimation by Cyanmethemoglobin method
When to Calibrate • At installation. • After the replacement of any component that involves dilution characteristics or the primary measurements (such as the apertures). • When advised to do so by your service representative
ICSH Reference methods PCV The reference PCV is Standard whole blood hemoglobin concentration (pack red cell hemoglobin concentration) microhematocrit centrifuge -The measurement on packed red cells is performed on cells obtained from the middle of the column of red cells RBC AND WBC TOTAL COUNT PLATELET a semi-automated single-channel aperture- flow cytometry using a fluorochrome – labelled impedance method with serial dilutions. monoclonal antibody, mixture of CD41, CD42a or CD61 (at least 2 antibodies)
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