HEML113 Week 1 PBF Prep & Staining 2024 Student PDF

Peripheral Blood Film Preparation & Wright’s Stain HEML113 FALL 2024 Competencies 2. Prepare and evaluate a Wright’s-stained peripheral blood film. Learning Objectives 2.a. Describe the samples, technique, sources of error, and criteria of an acceptable peripheral blood film....

Peripheral Blood Film Preparation & Wright’s Stain HEML113 FALL 2024 Competencies 2. Prepare and evaluate a Wright’s-stained peripheral blood film. Learning Objectives 2.a. Describe the samples, technique, sources of error, and criteria of an acceptable peripheral blood film. 2.b. Explain the principle, technique, and sources of error of the Wright stain for blood films. 2.c. Produce and critique a Wright’s-stained peripheral blood film. 2.d. Describe and follow safety policies with respect to working with biological samples, using lab equipment and waste disposal. 2.e. Describe the principle, advantages, and general use of automated slide stainers.  Keohane et al., 7th edition: ‒ Chapter 1 p. 1-7 ‒ Chapter 14 p. 225-230 Assigned Carr Atlas 6th ed.: Readings ‒ Chapter 1 p. 1-10 SOPs 1.1, 1.2, 3.1, & 4.1 What is Hematology? 1. Clinical study of blood cells and blood-forming tissues. Keohane EM, Walenga JM, Otto CN. Rodak's Hematology: Clinical Principles and Applications. St. Louis, Missouri.: Elsevier; 2020. 2. The diagnosis, treatment, and prevention of diseases of the blood and bone marrow as well as of the immunologic, hemostatic (blood clotting) and vascular systems. Because of the nature of blood, the science of hematology profoundly affects the understanding of many diseases. https://www.medicinenet.com/script/main/art.asp?articlekey=22594 What’s in Blood? What are the main components of blood? Plasma – liquid portion of blood which transports and nourishes the cells Cellular components – consist of the RBCs, WBCs, and PLTs What’s in Blood? What are the alternate names of each cellular component? WBC – Leukocytes Platelets – Thrombocytes RBC – Erythrocytes Keohane EM, Walenga JM, Otto CN. Rodak's Hematology: Clinical Principles and Applications. St. Louis, Missouri.: Elsevier; 2020:162. Hematology Department CBC testing – Whole blood Coagulation testing – Plasma Flow Cytometry Hematology & CBC Physicians rely on Hematology laboratory test results to diagnose and monitor therapy for diseases that affect blood cells ‒ By expertly staining, counting, analyzing and recording the appearance of all three cell types of cells (RBCs, WBCs, and PLTs) we can provide the physician with a Complete Blood Count (CBC) https://www.beckmancoulter.com/products/hematology/coulter-act-5diff-al  Physician orders a CBC Sample is collected Sample is run on automated cell counter Hematology Automated results are reported or a PBF is required Workflow Blood film is made on glass slide Blood film is stained Blood film is coverslipped Microscopic evaluation of PBF is preformed  Hematology Workflow Run CBC on Automated Analyzer Results within RI? Results Any analyzer Abnormal or May need flags? Questionable? Manual count Report CBC Make, stain and examine PBF for: RBC, PLT, WBC morph Perform Estimates Additional Results WBC Differential Reflex Valid? tests Acceptable Specimens for Peripheral Blood Films Venipuncture specimens – whole blood taken from veins (preferred sample) Capillary blood from finger-stick or heel-stick are acceptable – smears made at beside before blood clots Patient identification is essential ‒ Two identifiers minimum required ‒ VPML121 Sample Procurement Fall 2024 https://www.wikiwand.com/en/Vacutainer Acceptable Specimens for CBC & PBFs Sample must be anticoagulated ‒ Stops blood from clotting ‒ If sample clotted, cells are bound in the clot and cannot be assessed EDTA (lavender top tube) is the anticoagulant of choice for routine Hematology testing ‒ Filled to appropriate volume ‒ Must be immediately, gently and thoroughly mixed following collection EDTA Whole Blood Sample EDTA = Ethylenediaminetetraacetic acid Chelates calcium ‒ Calcium is necessary in the coagulation cascade- its removal inhibits a series of events which cause clotting Preserves cellular components and morphology (least amount of cell distortion) Collection tubes with EDTA should be mixed by 8-10 end-to-end inversions immediately following venipuncture collection Microcollection tubes with EDTA should be mixed by 10 complete end-to-end inversions immediately following collection Either collection tube must be mixed prior to analysis https://www.bd.com/en- ca/offerings/catalogue/product- search?search=EDTA Blood Stability, Storage & Retention EDTA specimens should be analyzed within six hours of collection at room temperature. Microcollection EDTA specimens should be analyzed within four hours of collection at room temperature. Blood smears should be prepared within four hours of collection to reduce cell deterioration and morphology artifacts. If testing cannot occur within required time restrictions, store at 4°C. Stability – 24 hours at room temperature. Retention – facility dependent.  Unacceptable Specimens for CBC & PBFs Partially or Under-Filled Tubes Blood : Anticoagulant ratio is important ‒ Too-little blood = too much EDTA Excess EDTA leads to: ‒ Erroneously low blood cell counts and Hematocrits ‒ Staining alteration ‒ Morphologic changes to RBCs and WBCs on blood film o RBC and WBC shrinkage https://jag.journalagent.com/ijmb/pdfs/IJMB-50876-TECHNICAL_REPORT-GUVEN.pdf o Crenated RBCs o WBC membrane damage Tube must be filled ± 10% of the stated draw volume Unacceptable Specimens for PBFs Overexposure to EDTA Recall, PBF should be made within 4 hours at RT Cells in EDTA > 4 hours at RT show artifacts: ‒ Crenated or spherocytic RBCs ‒ MCV – Can be increased due to RBC swelling ‒ HCT – Can be increased due to increases in MCV ‒ Necrobiotic/necrotic (dying) WBCs (decreasing counts) ‒ Progressively vacuolated WBCs (especially Neutrophils and Monocytes) ‒ Neutrophils – swelling, loss of granules and Pelger-Huet-type nuclear changes ‒ Eosinophils and Basophils – degranulation ‒ Large platelets Storage at 4C for up to 6 hours minimizes changes  RBC & WBCs Crenated RBCs (pie crust membrane) and Necrobiotic WBCs (we see nucleus Unacceptable fragmenting and shrinking) Specimens for PBFs Vacuolated Neuts Vacuoles are pale staining area – any clear space or cavity formed within the cytoplasm of a cell- very large and abnormal looking Normal Unacceptable Specimens for CBC & PBFs Over-Filled Tubes Would only occur if filling EDTA tube from a syringe Prevents proper mixing of sample = Insufficient EDTA for blood volume May lead to platelet clumping and clotting ‒ Clotted sample – Reject specimen Tube must be filled ± 10% of the stated draw volume Unacceptable Specimens for CBC & PBFs Incorrect Phlebotomy Technique ‒ Slow/difficult venipuncture draw, improperly mixed tubes or improper handling means blood may not get exposed to EDTA in time Small clots (fibrin strands or microclots) or clumping platelets may affect patient results You can’t tell this from the tube – you must make a PBF ‒ Automated platelet count may be unexpectedly (falsely) decreased Unacceptable Specimens for CBC & PBFs Clotting = criteria for Hematology specimen rejection Unacceptable Specimens for CBC & PBFs This EDTA sample is clotted It would be obvious when you picked up the tube This sample cannot be run and must be rejected A request will be made to recollect the sample https://twicsy.com/i/yVGexe Unacceptable Specimens for PBFs This is a clot taken out of an EDTA tube, which may have been missed prior to running the sample What most likely caused this clot formation? ‒ Too little sample? ‒ Too much sample? ‒ Poor mixing? ‒ Expired tube? https://www.beaumontlaboratory.com/docs/default-source/specimen-collections- manual/blood/1233298-prevent-clotting-with-anticoagulant- tubesa4cfeafcc28e4b7ca0d3d8d3d93bfce1.pdf? PBF Preparation Manual Wedge Technique Peripheral Blood Film Preparation Manual Wedge Technique: 2 slides required- beveled- edges 1 drop of well-mixed EDTA sample (about 2-3 mm in diameter) Hold spreader slide at a 30°- 45° angle Draw spreader slide back into drop of blood; blood will run along edge of spreader Quickly and smoothly push the spreader forward Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:203  Peripheral Blood Film Preparation Aid for Manual Wedge Technique: Blood dispenser Cuts the time of smear- making in half, reducing technician fatigue Semi-Automated Wedge Technique: A small mechanical device for blood smear preparation Offers high and consistent quality, reduces inter- operator variability Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:203 https://www.fishersci.com/us/en/browse/90226262/blood-smear-preparation-devices  Body of the smear – not too thin or thick – this is where we examine the cells ‘Feathered Edge’ or Head of the smear – tail at the thinnest edge too thick to see cells of the smear Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:203  Peripheral Blood Film Preparation Features of a well-made PBF: 1. Film is ⅔ – ¾ the length of the slide. 2. Film has slightly rounded or straight feathered edge (not bullet shaped). 3. Is slightly thinner at the sides than the slide itself (lateral edges of the film are visible). 4. Film has a smooth homogeneous appearance without irregularities, streaks, dark bands, or holes. 5. When held up to the light, the thin portion (feathered edge) has a ‘rainbow’ appearance. Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:203 6. The entire drop of blood is picked up and spread across the slide (no dried blood where drop originated). Unacceptable PBFs A. Chipped or rough edge on spreader slide. B. Hesitation in forward motion of spreader slide. C. Spreader slide pushed too quickly. D. Drop of blood too small. E. Drop of blood not allowed to spread across the width of the slide. F. Dirt or grease on the slide; may also be due to elevated lipids in the blood specimen. G. Uneven pressure on the spreader slide. H. Time delay; drop of blood began to dry. Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:204  Sources of Error & Troubleshooting Poor Technique a. The drop of blood is too large (long, thick smear), too small (short smear) or too much left behind (dried circle at end of smear) – adjust size of drop of blood accordingly. b. The spreader is pushed with a jerky motion and/or lifted off the slide (banding on smear) – hold spreader firmly against slide and use a smooth motion with even pressure. c. The spreader is not pushed rapidly enough (smear may be too long and larger WBC such as, monocytes and granulocytes, are pushed to the sides/end of smear) – adjust speed accordingly. d. The blood in the capillary tube has dried up and does not flow – need to ‘tap’ to get blood out – DISCARD capillary tube and use a new one. Sources of Error & Troubleshooting cont’d Poor Technique Thickness & length of the smear is influenced by the spreader angle: ‒ Smaller angle – longer, thinner smear ‒ Larger angle – shorter, thicker smear Troubleshooting: ‒ Smears too long – adjust angle of spreader up or use smaller drop (gives shorter, thin smear) ‒ Smears too short – adjust angle of spreader down or use larger drop (gives longer, thicker smear) Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:203 Sources of Error & Troubleshooting cont’d Equipment a. The slide is greasy or dirty (streaks or holes in the smear) – discard and use new slide. b. The spreader slide is chipped or rough (jagged edge to smear) – get a new spreader or check box of slides for damage. Sample a. Hemoglobin, Hematocrit or RBC count very low – smear is very long and thin. Adjust spreader angle up to shorten smear. b. Hemoglobin, Hematocrit or RBC count is high – smear is short and thick. Adjust spreader angle down to lengthen the smear. Drying Blood Films Best method – air dry slides as quickly as possible but without heat: Reduced air humidity Don’t blow on slides or wave slides around Lay slides flat or stand upright Slides must be completely dry before staining ‒ If not, head of smear will wash off during staining process or ‒ Artifacts may appear on smear Drying Blood Films Water or Drying artifact occurs if slides dry too slowly or too much moisture/humidity is present ‒ RBCs look ‘moth-eaten’ (holes) or crenated ‒ Central pallor of some RBCs are too distinct – ‘punched out’ artifact ‒ Refractive (shiny) rings on RBCs Extremely anemic samples – increased Plasma:RBC ratio ‒ Excess moisture – hard to avoid drying artifact Carr, JH and Rodak, B. Clinical Hematology Atlas. 5th ed. St. Louis, MI: Elsevier; 2017:213 PBF Staining Manual & Automated Wright’s Stain  Staining Peripheral Blood Films Aids the dentification and evaluation of normal or abnormal blood cell morphology Specifically stains cytoplasm, nucleus and other cellular components ‒ Demonstrates the shape and hemoglobin content in RBCs ‒ Differentiates the various types of WBCs ‒ Visualize PLTs Romanowsky stain is used for peripheral blood and bone marrow smears  Principle of Romanowsky Stains Romanowsky stains include: Wright Wright-Giemsa Jenner Leishman Romanowsky stains are “polychromatic” ‒ “Poly” = many or much ‒ “Chrom” = colour Range of colours between red and blue Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, Alcoholic stain MI: Elsevier; 2020:1. Principle of Romanowsky Stains Pure Wright stain or Wright-Giemsa are the most commonly used stains for PB and BM films ‒ They are considered ‘polychrome’ stains because they contain both Eosin (Y or B) & Methylene Blue Giemsa stain also contains Methylene Blue Azure ‒ Best for staining early RBCs with excess RNA Staining is carried out via manual or automated methods- same goal- consistent day-to-day staining quality is essential- dependent on lab and volume of slides to be stained/workload  Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:205. METHOD: Manual 1. Flood slide with filtered Wright Stain to fix cells Wright to glass slide (4 min) Staining – 2. Add equal amount of buffer to stain – mix until a green metallic sheen forms (8 min) Hematology 3. Rinse with light stream of tap water SOP 4.1 4. Wipe back of slide to remove any residue 5. Air dry  Wright’s Stain Procedure General Wright’s Stain Steps: 1. Add Stain (to fixed slide) 2. Add phosphate buffer @ pH 6.4 to the stain & mix 3. Rinse & Dry Methanol fixes the cells to the slide (added prior to staining especially if staining delayed or in the stain) Staining of the cellular components does not occur until buffer is added ‒ Staining reactions are pH dependent; the buffer should be at a pH of 6.4 – 6.8 to facilitate staining Free methylene blue and free eosin will then stain their target tissue components (acidic and basic cell components will either pick up methylene blue or eosin) Oxidized methylene blue and eosin form a thiazine-eosinate complex which stains the neutral components ` General Staining Principle ACID & BASIC DYES Dye attaches to ACID DYE BASIC DYE oppositely- Charge of Negative Positive charged tissue dye ion (Anionic) (Cationic) components Charge of Positive or Negative tissue Basic (+) or Acid (-) Tissue Acidophilic Basophilic described as: Principle of Free methylene blue is basic and stains acidic cellular components from blue-grey to Wright’s dark blue colour: ‒ i.e., Nucleic Acids: RNA & DNA, Stain nuclear and cytoplasmic proteins, and Basophil granules Carr, JH and Rodak, B. Clinical Hematology Atlas. 5th ed. St. Louis, MI: Elsevier; 2017:77. Principle of Free eosin is acidic and stains basic cellular components from light to deep Wright’s pink to red or orange colour: Stain ‒ i.e., Hemoglobin and Eosinophilic granules Carr, JH and Rodak, B. Clinical Hematology Atlas. 5th ed. St. Louis, MI: Elsevier; 2017:17, 71.  Neutrophils have cytoplasmic granules Principle of with neutral pH and pick up some staining Wright’s characteristics from both stains (thiazine- eosinate complex) Stain ‒ Neutrophil granules appear pink/violet (lavender to lilac) Carr, JH and Rodak, B. Clinical Hematology Atlas. 5th ed. St. Louis, MI: Elsevier; 2017:53, 71. Mechanism of Staining Ionic bonding and Buffer pH Dye charge does not change within pH range of staining methods Overall charge of tissue proteins can change with solution pH: ‒ Some protein molecules are amphoteric ‒ Addition of acid or base to solution can result in a change in the overall charge of the tissue Mechanism of Staining Ionic bonding and Buffer pH Add acid (H+) to solution = more +ve charges, more attachment sites for acid dye ‒ Stronger eosin staining – RBCs too red ‒ Weaker methylene blue staining – WBCs too pale Add base (OH-) to solution = more -ve charges, more attachment sites for basic dye ‒ Stronger methylene blue staining – nuclei very dark ‒ Weaker eosin staining – RBCs & Eosinophils look grey Buffer pH must be between 6.4 to 6.8  Staining Patterns of Hematological Cells Pink: Dark Purple-Black: Neutrophil cytoplasm Basophilic granules RBCs (HGB- pink to salmon) Eosinophil granules (can be red to Blue: orange as well) Nuclei (dark) Pink-Violet: Lymphocyte cytoplasm Neutrophilic granules Platelet granules Grey-Blue: Monocyte cytoplasm Purple: Nuclei (dark) Purple or Red: Azurophilic granules  Staining of RBCs & PLTs https://www.quora.com/What- is-the-shape-of-a-red-blood-cell You can see the central pallor in the RBCs. RBCs (HGB) stains a salmon colour. The platelet cytoplasm stains light blue to colourless with abundant red to violet granules. Staining of The 5 Basic White Cells Neutrophil Lymphocyte Eosinophil Basophil Monocyte Carr, JH and Rodak, B. Clinical Hematology Atlas. 5th ed. St. Louis, MI: Elsevier; 2017. Summary: Features of a Optimally-Stained PBF Macroscopically - ‒ Smear should appear pink to purple Microscopically – 1. RBCs are pink to salmon 2. Nuclei are dark blue to purple 3. Neutrophil cytoplasmic granules are lavender to lilac 4. Eosinophil cytoplasmic granules are red to orange 5. Basophil cytoplasmic granules are dark purple to black 6. Area between cells should be colourless, clean, and free of stain precipitates  Troubleshooting PBF Staining PROBLEM CAUSE & RESOLUTION Stain precipitates on slide Filtering of stain required Insufficient rinsing (see SOP) 1 Allowing stain to dry on slides during staining process RBCs appear grey (brown Stain or buffer too alkaline (most or green) common) Inadequate rinsing 2 Eosinophil granules are Prolonged staining grey, not orange Blood film too thick Heparinized blood sample WBC nuclei are too dark RBCs red colour Stain or buffer too acidic (most common) WBC nuclei barely visible Under-buffering (too short time) 3 Over-rinsing Blood film too thin Stain old Troubleshooting PBF Staining Stain deposit ‒ Stain needs to be filtered ‒ Poor rinsing of slide ‒ Stain dried on slide https://veteriankey.com/peripheral-blood-smears-2/ Troubleshooting PBF Staining Inadequate staining – WBC nuclei very pale ‒ Stain or buffer too acidic ‒ Insufficient time in buffer (under-buffer) ‒ Over-rinsing ‒ Stain is too old https://veteriankey.com/peripheral-blood-smears-2/ Troubleshooting PBF Prep/Stain What is the issue? https://veteriankey.com/peripheral-blood-smears-2/ Troubleshooting PBF Prep/Stain What is the issue? https://veteriankey.com/peripheral-blood-smears-2/ Troubleshooting PBF Prep/Stain What is the issue?  Automated Slide Stainers Hematek Benchtop Stainer Holds 25 slides at once Continuous feed – 1 min https://www.dotmed.com/listing/slide-stainer/siemens/hematek-2000-slide-stainer-4488c-hematek/2622060 between slides 10 minutes to stain Add slides to conveyor by hand Easy to use & walk away Uniform staining Little maintenance https://www.siemens-healthineers.com/hematology/systems/hematek-3000  Automated Slide Stainers https://www.fishersci.com/shop/products/siemens-diagnostics-modified-wright-giemsa-stain-pack-hema-tek-2000-slide- stainer/23044626 https://www.dotmed.com/listing/slide-stainer/siemens/hematek-2000-slide-stainer-4488c-hematek/2622060  Automated Slide Maker and Stainer Part of a fully automated Hematology analyzer One sample aspiration for CBC & slide production Criteria for PBF review are built into Beckman Coulter LH 750 system and when met, slide prepared https://www.beckmancoulter.com/download/file/wsr-32948/4277248DD?type=pdf Wedge preparation considers patient hematocrit reading (determines size of drop of blood) Slide is stained then dried and ready for evaluation Sysmex SP-10 Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:204. Quick Stains https://www.medicine.mcgill.ca/physio/vlab/bloodlab/diff_count_practice.htm Commercially prepared, all-in-one Wright or Wright- Giemsa stain Approximately one minute stain procedure Convenient and cost effective for low-volume laboratories Used when rapid Turn-Around-Time (TAT) required Quality is often a concern, but timing can be slightly adjusted to attain quality colour Coverslipping Slides Good-quality glass cover slips are used to cover stained PBFs Held in place by ‘mounting media’ – ‒ Synthetic resins, such as, Permount Protect smear while stored https://www.thermofisher.com/order/catalog/product/24X30-1W- Minimize biological hazard YO#/24X30-1W-YO Microscopes are designed to focus on slides with coverslips – slide & coverslip have same RI ‒ Objectives show maximum coverslip thickness (in mm) Essential to set up microscope for optimal slide viewing (Kohler Illumination) https://www.microscopyu.com/microscopy-basics/microscope-objective- specifications Next Week… Lecture Week 2 Examination of PBF & Cellular Components of Blood- Leukocyte Morphology ‒ Review presentation, learning objectives and assigned readings listed in first few slides of posted presentation Lab Week 2 Introduction to the Compound Light Microscope – Parts, Use & Ergonomics Peripheral Blood Film Preparation, Staining & Intro to Blood Film Examination Next Week… Lab Week 2 First ONSITE lab – Room 1025 See Seating Plan for bench assignment (posted on Blackboard and will also be posted outside of lab) Review Microscopy Tutorial material from Week #1 ‒ MUST review DEMO VIDEOS for preparing PBFs and for staining (manual and automated) ‒ No demonstrations during labs, only practice Week 2 Lab Exercises ‒ Review exercises and prerequisite readings ‒ Printed copy will be provided – bring a binder to store your exercises for the semester Bring your lab coat, face shield, Atlas, binder for your lab exercises and writing implements Remember to wear lab-appropriate clothing and shoes Arrive 15 min early (if not coming from another lab) and come prepared! References Carr, JH. Clinical Hematology Atlas. 6th ed. St. Louis, MI: Elsevier; 2022:1–10. Keohane, EM, Otto, CN, and Walenga, JM. Rodak’s Hematology: Clinical Principles and Applications. 6th ed. St. Louis, MI: Elsevier; 2020:1-7, 201-217. McCall RE, Tankersley CM. Phlebotomy Essentials. 6th ed. Philadelphia, PA: Wolters Kluwer; 2016: 179-208, 283-284.