Introduction to Clinical Laboratory Hematology PDF

INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 1 HEMATOLOGY The study of blood cells and coagulation. It includes: the analyses of the concentration, structure, and function of cells in the blood; their precursors in the bon...

INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 1 HEMATOLOGY The study of blood cells and coagulation. It includes: the analyses of the concentration, structure, and function of cells in the blood; their precursors in the bone marrow; chemical constituents of plasma or serum intimately linked with blood cell structure and function; and functions of platelets and proteins involved in blood coagulation. (McPherson, et. Al, 2007)  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 2 GENERAL CHARACTERISTICS OF BLOOD It is a liquid Connective Tissue https://c8.alamy.com/comp/A78YMW/sem-x4000-blood-clot- A78YMW.jpg  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 3 GENERAL CHARACTERISTICS OF BLOOD An average human possesses approximately five (5) liters of blood representing 6-8% of total body weight. https://basicmedicalkey.com/wp- content/uploads/2016/05/F500323f20-01-9780323096003.jpg  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 4 GENERAL CHARACTERISTICS OF BLOOD https://qph.cf2.quoracdn.net/main-qimg-a65a19e9518d0712c96c1a5277070241.webp Blood pH ranges between 7.35-7.45  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 5 BRIEF HISTORY OF HEMATOLOGY Athanasius Kircher described “worms” in blood (1657) Anton Van Leeuwenhoek (1674) made an account of blood Guilio Bizzozero (Late 1800) described platelets as “Petites Plaques” James Homer Wright (1902): developed staining technique that improved visualization of blood cells https://www.researchgate.net/publication/324484207/figure/fig2/AS:881083243585537@1587078023331/Anto nie-van-Leeuwenhoek-and-an-example-of-his-hand-lens-microscopes-Courtesy-of-Wellcome.jpg  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 6 COMPOSITION OF BLOOD 55 % PLASMA 45 % FORMED ELEMENTS  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 7 COMPOSITION OF PLASMA 55 % 91% Water 7% Protein Other Nutrients PLASMA Waste Products  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 8 COMPOSITION OF FORMED ELEMENTS Platelets and Leucocytes 45 % FORMED ELEMENTS Erythrocytes  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 9 FORMED ELEMENTS OF BLOOD Erythrocytes Anucleated, organelle-free, bi-concave disc shaped cells required for gaseous exchange Leukocytes Immune cells that are categorized into granulocytes (neutrophil, basophil and eosinophils) & agranulocytes (monocytes and lymphocytes) Platelets: Cellular fragments that play a major role in blood hemostasis through preventing excessive blood loss  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 10 SERUM VERSUS PLASMA Serum is blood plasms minus the Plasma contains all fibrinogen coagulation proteins Extracted from blood after including fibrinogen clotting Extracted from blood without the need for clotting  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 11 GENERAL FUNCTIONS OF BLOOD Supply oxygen and nutrients to cells Remove carbon dioxide and other waste from cells Regulate body temperature and pH Body defense (Immunity) Coagulation Transport of regulatory molecules (Hormones, Cytokines)  INTRODUCTION TO CLINICAL LABORATORY HEMATOLOGY 12 HEMATOPOIESIS Process of blood cell production and maturation Major site in adults: Red Bone Marrow  LABORATORY SAFETY 13 INTRODUCTION Every laboratory worker should be aware of the potential hazards in their workplace and of importance of safety of their practice from specimen collection to waste disposal.  LABORATORY SAFETY 14 BIOLOGICAL HAZARDS “Biohazards” Anything harmful or potentially harmful to health. Body fluids (blood, urine & spinal fluid) may contain highly infectious & potentially lethal organisms. Extreme caution should be taken in collection, handling & processing all body fluids.  LABORATORY SAFETY 15 BIOLOGICAL HAZARDS Biohazards cause the great concern of all hazards in hematology laboratory. Due to the nature of the specimen analyzed in the lab: BLOOD Blood may contain highly infectious agents: viruses that cause hepatitis & AIDS. As infectious agent may be present in specimen for long time before signs & symptoms appear on patient, all biologic specimens should be considered biohazardous.  LABORATORY SAFETY 16 BIOHAZARDS EXPOSURE ROUTES AIRBORNE Biohazards can become airborne and inhaled when splashes, aerosols, or fumes are generated. Centrifugation Removal of tube stoppers Preparation of specimen aliquots  LABORATORY SAFETY 17 BIOHAZARDS EXPOSURE ROUTES INGESTION Biohazards can be ingested if healthcare workers neglect to sanitize hands before handling food, gum, candy, cigarettes, or drinks. Can also happen when workers cover their mouth with hands instead of tissue when coughing or sneezing, biting nails, chewing on pens or pencils, and licking fingers  LABORATORY SAFETY 18 BIOHAZARDS EXPOSURE ROUTES NON-INTACT SKIN Biohazards can enter the body through visible and invisible pre-existing breaks in the skin such as abrasions, burns, cuts, scratches, sores, dermatitis, and chapped skin.  LABORATORY SAFETY 19 BIOHAZARDS EXPOSURE ROUTES PERCUTANEOUS Through the skin Exposure to biohazardous microorganisms in blood or body fluid occurs through intact (unbroken) skin as a result of accidental needlesticks and injuries from other sharps including broken glass and specimen tubes.  LABORATORY SAFETY 20 BIOHAZARDS EXPOSURE ROUTES PERMUCOSAL Through mucous membranes Exposure occurs when infectious microorganisms and other biohazards enter the body through the mucous membranes of the mouth and nose and the conjunctiva of the eyes in droplets generated by sneezing or coughing, splashes, and aerosols and by rubbing or touching the eyes, nose, or mouth with contaminated hands.  LABORATORY SAFETY 21 GENERAL SAFETY RULES Personal protective equipment (PPE): Must be worn when handling biologic specimens. PPE includes: a) Laboratory coat b) Gloves c) Face shields (e.g., masks & plastic shields). d) Closed shoes.  LABORATORY SAFETY 22 GENERAL SAFETY RULES Laboratory coats or gowns: long sleeved, tight-fitting cuffs, buttoned up & clean Frequent washing is mandatory Should never be worn outside laboratory  LABORATORY SAFETY 23 GENERAL SAFETY RULES Gloves: Disposable gloves are worn in the following cases: ✓ When there is contact with blood or any body fluid. ✓ When venipuncture or finger sticks are performed Disposable Gloves: They must be changed in the following cases: 1. After each contact with a patient. 2. When there is visible contamination. 3. If physical damage occurs. They are not washed, reused and they should not be worn outside the lab. Gloves must be removed on completion of laboratory tasks, when using a telephone, or when performing any office work.  LABORATORY SAFETY 24 GENERAL SAFETY RULES Proper Gloves Removal Technique(Sliding Method)  LABORATORY SAFETY 25 GENERAL SAFETY RULES Eyewear (face shields and masks): Eyewear is worn when there is potential for aerosol splashes or sprays to mucus membranes (mouth, eye and nose) such as: 1. Removing caps from specimens. 2. Working at the cell counter. 3. Centrifuging specimens.  LABORATORY SAFETY 26 GENERAL SAFETY RULES Hand washing: Washing with water & soap is recommended. ✓After removing gloves. ✓Contamination with blood or body fluids. ✓When work is completed. ✓Before leaving the laboratory.  LABORATORY SAFETY 27 GENERAL SAFETY RULES Proper Hand Washing Technique  LABORATORY SAFETY 28 GENERAL SAFETY RULES Don’t remove specimen tube stoppers until necessary: Centrifugation should be performed with stoppers in place. A clear shield between laboratorian & the blood sample being manipulated could be used.  LABORATORY SAFETY 29 GENERAL SAFETY RULES Obtain immediate treatment of accidental & inappropriate contact with biohazards: Any contamination of skin cuts or needle punctures should be reported immediately to the supervisor & appropriate precaution will be taken.  LABORATORY SAFETY 30 GENERAL SAFETY RULES Hepatitis B virus vaccination should be given to all lab workers either before or within 10 days of starting work in the laboratory.  LABORATORY SAFETY 31 GENERAL SAFETY RULES Disposables: Properly dispose contaminated lab supplies: Medical wastes: (yellow/ red bags) 1. Any material contaminated with blood (e.g., gloves, tissues & cotton)→ should be disposed in biohazard bags. 2. Sharps (e.g., Needles, Glass slides, Pipette tips & Lancets)→ should be disposed in puncture-resistant containers (sharps container):  LABORATORY SAFETY 32 GENERAL SAFETY RULES Used disposable needles must not be bent, sheared, broken, recapped or removed from disposable syringes, rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and a dustpan, tongs, or forceps.  LABORATORY SAFETY 33 GENERAL SAFETY RULES Other general rules: ✓Mouth pipetting is strictly prohibited ✓Use Pasteur pipettes ✓Mechanical pipetting, not mouth pipetting, must be used for the manipulation of all liquids in the laboratory.  LABORATORY SAFETY 34 GENERAL SAFETY RULES ✓Domestic wastes:(white / black color bags). Personal wastes (e.g., papers & tissues). Disposed in domestic garbage  LABORATORY SAFETY 35 GENERAL SAFETY RULES Disinfection & cleaning of work surfaces: Work surfaces must be decontaminated with a chlorine solution (e.g. bleach) routinely at the completion of work and following any spill of potentially infectious material  LABORATORY SAFETY 36 GENERAL SAFETY RULES Never touch, taste, or smell any chemical that you do not know for a fact is harmless. Food and drink must not be kept in the same refrigerator as laboratory specimens or reagents or where potentially infectious materials are stored or tested. When performing a lab, make sure the work area has been cleared of bags, books, jackets…..etc.  LABORATORY SAFETY 37 GENERAL SAFETY RULES All of the following are prohibited in laboratory: Eating. Drinking. Smoking. Chewing gums Untied long hair.  Introduction to the Clinical Hematology Laboratory 38 THE HEMATOLOGY SECTION OF THE LAB The hematology laboratory is integral to patient diagnosis, treatment, and overall healthcare, providing critical information that guides clinical decision- making and supports the broader medical community in improving patient outcomes.  Introduction to the Clinical Hematology Laboratory 39 ROUTINE HEMATOLOGIC TESTS Complete Blood Count Total RBC count Hematocrit (hct) or Packed Cell Volume (PCV) Hemoglobin RBC Indices MCV MCH MCHC Red Cell Distribution Width Total WBC count Differential Count Platelet Count  Introduction to the Clinical Hematology Laboratory 40 COMPLETE BLOOD COUNT RED CELL PARAMETERS Total RBC count Hematocrit v Hemoglobin RDW RBC Indices v  Introduction to the Clinical Hematology Laboratory 41 COMPLETE BLOOD COUNT WHITE BLOOD CELL PARAMETERS Total WBC count Differential Count Relative Count of each WBC type Absolute Count of each WBC type https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%2Ffigure%2FMorphological-classes-of-leukocytes-a-Eosinophil-b-Lymphocyte-c-Monocyte- d_fig1_343955468&psig=AOvVaw3QLh6lmyvFKmT5NO9V4hWZ&ust=1724700692827000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCKCpq_PwkIgDFQAAAAAdA AAAABAE  Introduction to the Clinical Hematology Laboratory 42 COMPLETE BLOOD COUNT PLATELET PARAMETERS Total Platelet Count Mean Platelet Volume This Photo by Unknown Author is licensed under CC BY-NC-ND  Introduction to the Clinical Hematology Laboratory 43 PROCEDURES USED IN THE PROCUREMENT & DOCUMENTATION OF SAMPLES RECEIVED IN THE HEMATOLOGY LABORATORY All patient samples received in the laboratory for hematology testing must be accompanied by doctor’s request  Introduction to the Clinical Hematology Laboratory 44 PROCEDURES USED IN THE PROCUREMENT & DOCUMENTATION OF SAMPLES RECEIVED IN THE HEMATOLOGY LABORATORY Most samples are venous collections & arrive to the lab from the wards or outside clinics within 12 hours of collection. Samples should be received in a sealed, clear, clean plastic bag  Introduction to the Clinical Hematology Laboratory 45 PROCEDURES USED IN THE PROCUREMENT & DOCUMENTATION OF SAMPLES RECEIVED IN THE HEMATOLOGY LABORATORY Both the samples & paperwork must match & be correctly identified with the patients full name, D.O.B. & health card number. The request form must indicate the tests to be performed https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.shutterstock.com%2Fimage-photo%2Fblood- testing-patient-on-test-request- 2226780895&psig=AOvVaw3T6qyGRcL0LVDclXqOF7_h&ust=1724701446023000&source=images&cd=vfe& opi=89978449&ved=0CBQQjRxqFwoTCMjSuNvzkIgDFQAAAAAdAAAAABAE  Introduction to the Clinical Hematology Laboratory 46 SPECIAL TESTS IN HEMATOLOGY RETICULOCYTE COUNT Immature erythrocytes Slightly larger than mature erythrocytes Contain remnants of ribosomal RNA Stained by supravital stains New Methylene Blue Brilliant Cresyl Blue Acridine orange https://www.google.com/url?sa=i&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FSupravital_staini ng&psig=AOvVaw22tYd2iNYN8hNrrGdMot46&ust=1724701809638000&source=images&cd=vfe&opi= 89978449&ved=0CBQQjRxqFwoTCPjanYj1kIgDFQAAAAAdAAAAABAR  Introduction to the Clinical Hematology Laboratory 47 SPECIAL TESTS IN HEMATOLOGY PERIPHERAL BLOOD SMEAR EXAMINATION Cell morphology Anisocytosis Poikilocytes https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%2Ffigure%2FAnisocytosis-poikilocytosis-and- hypochromia-of-erythrocytes-in-peripheral- blood_fig6_359452345&psig=AOvVaw1pn_Tp37ye0VpL69LoA4Tk&ust=1724701919801000&source=images&cd=vfe&opi=89 978449&ved=0CBQQjRxqFwoTCNiEsb_1kIgDFQAAAAAdAAAAABAE  Introduction to the Clinical Hematology Laboratory 48 SPECIAL TESTS IN HEMATOLOGY G6PD Testing Part of Newborn Screening Detects Inborn errors of metabolism https://www.google.com/url?sa=i&url=https%3A%2F%2Fgenetech.co.in%2Fnewborn- screening%2F&psig=AOvVaw2hgqD5s- bU4Bmo8hIo8sDT&ust=1724702325489000&source=images&cd=vfe&opi=89978449&ved=0C BQQjRxqFwoTCKiMxcz4kIgDFQAAAAAdAAAAABAE  Introduction to the Clinical Hematology Laboratory 49 SPECIAL TESTS IN HEMATOLOGY ERYTHROCYTE SEDIMENTATION RATE Measures the rate of settling of erythrocytes in an hour. Measure of inflammatory proteins in blood sample https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%2Ffigure%2FA- capillary-tube-mounted-with-anticoagulated-blood-for-checking-micro- ESR_fig1_372066662&psig=AOvVaw04fCf3QgzyuLLCavca7zn8&ust=1724702608887000&sour ce=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCKj5yYf4kIgDFQAAAAAdAAAAABAY  Introduction to the Clinical Hematology Laboratory 50 PRIMARY FUNCTIONS OF THE HEMATOLOGY LABORATORY Diagnosis of blood disorders Monitor treatment efficacy Blood count analysis as baseline data Assist in Transfusion medicine by conducting blood grouping Provide crucial data to detect infections and immune responses Research and development Support public health  Specimen Collection I 51 PRE-ANALYTICS Comprises all the processes that occurs before the laboratory analysis. Includes the indication, informing and identifying the patient, sample collection with subsequent transport and storage until centrifugation and sample distribution. https://www.google.com/url?sa=i&url=https%3A%2F%2Fcmpt.ca%2Fextending-the-scope-of-eqa-to-the-extra- analytical- phases%2F&psig=AOvVaw1K_cukJqqUUbt5gTbAmZ3J&ust=1724750092157000&source=images&cd=vfe&opi=8 9978449&ved=0CBQQjRxqFwoTCNiQ9f-okogDFQAAAAAdAAAAABAZ  Specimen Collection I 52 PRINCIPLES OF PREANALYTICS On average, the pre-analytical phase accounts for about 57% of the entire process between the patient and the analysis result (Guder et.al, 2009) About 25% of errors in pre- analytics have consequences for the patient. The test result can only be as good as is permitted by the patient sample obtained. https://www.google.com/url?sa=i&url=https%3A%2F%2Fskilldeer.com%2Fuae%2Fdubai%2Fbusiness- career%2Fhealthcare-professionals%2Fdiploma-in-phlebotomy-technician-bur-dubai- 9673&psig=AOvVaw3yiTsGZyTw5vEPK2k7cJJB&ust=1724750373221000&source=images&cd=vfe&o pi=89978449&ved=0CBQQjRxqFwoTCNj48_-pkogDFQAAAAAdAAAAABAE  Specimen Collection I 53 COMMON PREANALYTICAL ERRORS Hemolysis (44%) Underfilling (17%) Blood Clot (8%) Bonini et.al; Errors in Laboratory Medicine; Clin Chem 48:5; 691-698(2002) https://www.google.com/url?sa=i&url=https%3A%2F%2Fwelzo.com%2Fblogs%2Fblood-tests%2Fwhat-does-a-haemolysed-blood-test-result- mean&psig=AOvVaw2x1hd_amczIKyh7eF5GXce&ust=1724750530908000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCMC32seqkogDFQAAAAAdAAAAABAE https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%2Fpost%2FWhy-serum-was-clotted-after-separation&psig=AOvVaw3UmXANuGJQrKBgO4Pf-oj- &ust=1724750755038000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNDIvLmrkogDFQAAAAAdAAAAABAV https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.cliniciansbrief.com%2Farticle%2Ftop-5-blood-collection-sampling-errors&psig=AOvVaw3VNvXf- DY4_8FmdsPmP1Ic&ust=1724750629552000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCIiFsoGrkogDFQAAAAAdAAAAABAZ  Specimen Collection I 54 INTERFERING VS. INFLUENCING FACTORS INTERFERING FACTORS INFLUENCING FACTORS Alter test results and cause Change the concentration disruptions, depending on method of analytes which depend used. on medical condition and By changing the test method it must be considered when may be possible to eliminate evaluating results (Either interfering factors increased or decreased the Classified as results) Endogenous (internal); within the sample as a reflection of patient’s Classified as illness or condition Modifiable Exogenous (External): outside the Non-Modifiable patient sample  Specimen Collection I 55 INTERFERING VS. INFLUENCING FACTORS INTERFERING FACTORS INFLUENCING FACTORS Examples Examples Modifiable Drug use Endogenous Substance Use: Alcohol Spherocytosis (hemolysis) Substance Use: Nicotine Substance Use: Caffeine Hematocrit >65% (Elevation Medication in PTT and aPTT) Physical activity Exogenous Body Position Diet Medication (False Non-modifiable measurements) Race Gender Anticoagulants (False Pregnancy measurements) Age Cycling (Can increase PSA Biorhythm value) Circadian rhythm  Specimen Collection I 56 RESPONSIBILITIES OF THE PHLEBOTOMIST Organisation of the Blood Collection Correct documentation (Patient ID and time of Day) Instructing and preparing the patient for the sample collection Preparation of the sample (Centrifugation, if necessary) Storage until collection (by the appropriate section of the lab)  Specimen Collection I 57 PATIENT PREPARATION PATIENT PREPARATION 1. Check patients Identity. Request form Name (first, second & third name). Date of birth DOB 2. Proper sample identification Read request form carefully Decide how much blood & which tubes required. Check preconditions ie. Fasting.  PHLEBOTOMY / VENIPUNCTURE Venipuncture may be carried out using: 1. Syringe (Open System) 2. Evacuated tube system Syringe (Open System) Method (Vacutainer System) The most commonly used system for collecting blood Closed system: patient's blood goes directly from vein into a stoppered tube without being exposed to air. Numerous tubes with different types collected using a single Evacuated (Closed System) Tube System venipuncture Safer than using needle & syringe 3. Monovette System Combines the advantages of the syringe and evacuated system S-Monovette System SYRINGE (OPEN SYSTEM) Sterile, disposable & for single use only. Composed of: 1. End of needle inserted into the vein is “bevel” 2. Long, cylindrical portion is “shaft” 3. End connects to blood drawing apparatus is “hub”. The gauge “size” of needle is→ diameter of the lumen or "bore" of the needle. The larger the gauge number, the smaller the actual diameter of the needle. 20-25 gauge→ 21 gauge needles most commonly used Components of the Syringe Assembly 61 9/30/2024  VENIPUNCTURE 62 EVACUATED (CLOSED) SYSTEM Two-way needle Evacuated System Evacuated Tube  VENIPUNCTURE 63 EVACUATED (CLOSED) SYSTEM Evacuated Needle Assembly Needle Assembly with Needle Guard Evacuated Tube with Luer Lock with two-way needle attached to tube adapter Needle  Venipuncture using Evacuated System 1) Needle: Combined needle & valve. The end of needle consists of a rubber-sheathed valve. This is inserted into the “holder “an open-ended plastic cylinder” & is screwed into place. The needle can then be unsheathed & inserted into the vein. 2) Specimen tube (vacutainer/ evacuated tube) Has a vacuum within it→ Correct amount of blood is drawn into the tube. The cap of the vacutainer tube has a rubber seal. Vacutainer tube is placed into the holder & pushed on to the needle. Full tube can then be removed & another tube put on to the needle & valve.  VENIPUNCTURE EQUIPMENT 65 S-MONOVETTE SYSTEM Combines the advantages of the syringe method and evacuated system Can be operated as syringe or as evacuated system Vacuum is created by pulling the plunger until a click sound is heard and separating the plunger from the needle assesmbly Special types of needles allow multiple blood cell collection  VENIPUNCTURE EQUIPMENT 66 NEEDLES FOR S-MONOVETTE S-MONOVETTE SAFETY MULTIFLY NEEDLE S-MONOVETTE SAFETY NEEDLE ASPIRATION  Specimen Collection I 67 SPECIMEN TUBES / CONTAINERS ANTICOAGULANTS Evacuated tubes Anticoagulants are chemical substances that stop the blood Stopper /top clotting. If blood is collected into a plain tube, it will clot. Then, the specimen can be centrifuged. The top layer “serum” will be used for a Different anticoagulants are used in the collection number of lab tests. of blood depending on what tests are required. However, for a number of tests, it There is an international color-coding system in place to more easily identify tubes. is necessary to prevent blood clotting by collecting blood into Depending on the collection system, the cap will tubes containing an anticoagulant. be either a hemogard closure (not to be opened) or a traditional stopper. PURPLE TOP Contains the anticoagulant EDTA (Ethylene Diamine Tetraacetic Acid) Obtained as either the disodium salt or dipotassium salt “which is the internationally recommended anticoagulant”. Mechanism: EDTA works by binding with calcium in the blood This binding action is called chelation. Calcium is necessary for blood clotting. When calcium is removed, blood cannot clot. https://www.google.com/url?sa=i&url=https%3A%2F%2Fevenmedical.en.made-in-china.com%2Fproduct%2FuXiELISPaDUl%2FChina- Yellow-Gel-Clot-Activator-Tube-Blood-Vacuum-Collection- Tube.html&psig=AOvVaw0VXtbSrMtm0_VdF5mQHkmw&ust=1630411452246000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCPCUo obb2PICFQAAAAAdAAAAABAJ EDTA TUBE EDTA maintains the structure of both RBCs & WBCs & stops the clumping of platelets. Routine hematology: ✓CBC→ to count RBCs, WBCs & platelets ✓Blood smears to look at blood cells morphology ✓Hb A1C, reticulocyte counts, sickle cell, G6PD, Hb electrophoresis & blood grouping. EDTA can’t be used for special Hematology: 1. Coagulation tests→ it destroys the less stable clotting factors in the blood 2. Platelet function studies→ It inhibits platelet function https://www.google.com/url?sa=i&url=https%3A%2F%2Fevenmedical.en.made-in-china.com%2Fproduct%2FuXiELISPaDUl%2FChina- Yellow-Gel-Clot-Activator-Tube-Blood-Vacuum-Collection- Tube.html&psig=AOvVaw0VXtbSrMtm0_VdF5mQHkmw&ust=1630411452246000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCP CUoobb2PICFQAAAAAdAAAAABAJ EDTA TUBE The concentration is 1.5 +/- 0.25 mg/ ml of blood. Effects of excess EDTA: >2 mg/ ml blood, all blood cells are affected: 1. RBCs shrink in size. 2. Packed cell volume (PCV) ↓. 3. Mean cell hemoglobin concentration (MCHC) ↑. 4. WBCs structure is affected. 5. Platelets counts: ↑falsely high; As they swell & break up. The fragments may be big enough to be counted as normal platelets. So, make sure correct amount of blood is added to the sample tubes. Then, tubes must be thoroughly mixed by https://www.google.com/url?sa=i&url=https%3A%2F%2Fevenmedical.en.made-in-china.com%2Fproduct%2FuXiELISPaDUl%2FChina- inversion Yellow-Gel-Clot-Activator-Tube-Blood-Vacuum-Collection- Tube.html&psig=AOvVaw0VXtbSrMtm0_VdF5mQHkmw&ust=1630411452246000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCP CUoobb2PICFQAAAAAdAAAAABAJ BLUE TOP Trisodium citrate (32 g/l) → (9:1) Nine volumes of blood are added to 1 volume of sodium citrate solution. Then well- mixed. Mechanism: stops blood from clotting by binding calcium. So, both EDTA & citrate→ “chelating agents” Used for: 1) Coagulation studies & platelet function https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.indiamart.com%2Fproddetail%2Fvacuum-blood- collection-tubes-sodium-citrate-20659939933.html&psig=AOvVaw2V6Tk3- tests xoG0HYieuHXobJV&ust=1630411834013000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCPDpk8fb2PICFQ AAAAAdAAAAABAK Citrate preserves the clotting factors & platelet function. TRISODIUM CITRATE Trisodium citrate is also used to measure Erythrocyte Sedimentation Rate (ESR). (black stopper): For this test, 4 volumes of blood are added to 1 volume of citrate (4:1) Not used for→ CBC (measurement of red cell, white cell & platelet counts) It is in liquid form & dilutes blood. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.ec21.com%2Fproduct-details%2F3.8-Sodium-Citrate- ESR--10727912.html&psig=AOvVaw1kW8uVAqypeI_bN- g3ZUYD&ust=1630412063274000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCOD1q7Lc2PICFQAAAAAdAAAA ABAj GREEN TOP Used at a concentration: 1- 5 mg/ ml blood (10-50 IU) Mechanism: Used for: 1. Red cell osmotic fragility tests & red cell enzymes→ does not change the size of RBCs. 2. Testing arterial blood gases, electrolytes & Chromosomal studies Not used for: 1. Counting platelets & WBCs (CBC)→ https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.diytrade.com%2Fchina%2F because it causes clumping of the cells. pd%2F10041657%2FHeparin_Tube_Heparin_lithium.html&psig=AOvVaw2c9j8QQl5NYRJT cWzlW3- X&ust=1630413235936000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCLC- 2. Preparing peripheral blood films→ because it gives a faint blue lqTh2PICFQAAAAAdAAAAABAD background color GREY TOP Top color: Grey Potassium Oxalate is weak anticoagulant Fluoride stops glycolysis. “stabilizer” Used to” measure blood glucose level→ Chemistry department https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.hensomed.com%2Fproducts%2Fflu oride-glucose-tube%2F&psig=AOvVaw2g0iRhMrx- 9MHT4o7_bKLO&ust=1630413282924000&source=images&cd=vfe&ved=0CAsQjRxqFwoTCMC94 fjg2PICFQAAAAAdAAAAABAD PLAIN TUBES No anticoagulants inside them Red or yellow cap: Serum Separating Tubes Yellow: with Gel Separator Additives: Gel + Clot Activator Used for: Chemistry, Immunology & Blood bank  Specimen Collection 2 77 LEARNING OBJECTIVES List the equipment and supplies needed to collect blood specimens by venipuncture. Describe the purpose of the equipment and supplies needed to collect blood specimens by venipuncture. Explain the purpose / use of the equipment and supplies needed to collect blood specimens by venipuncture. List the components of the different techniques in performing venipuncture. Explain how each technique of performing venipuncture works  Specimen Collection 2 78 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Phlebotomy Chair should be comfortable for the patient and have adjustable armrests to achieve proper positioning of either arm. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.amazon.ae%2FHelsevesen-Comfortable-Drawing-Phlebotomy- Adjustable%2Fdp%2FB07R8ZRKYK&psig=AOvVaw0dq_AYQYKfTrrfWHVe0gaI&ust=1725335816520000&source=images&cd=vfe&opi=89978 449&ved=0CBQQjRxqFwoTCICYifyuo4gDFQAAAAAdAAAAABAc https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.labconco.com%2Fproduct%2Fblood-drawing- chairs%2F983&psig=AOvVaw0dq_AYQYKfTrrfWHVe0gaI&ust=1725335816520000&source=images&cd=vfe &opi=89978449&ved=0CBQQjRxqFwoTCICYifyuo4gDFQAAAAAdAAAAABAX https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.mistrymedical.com%2Fplinth- medical-reclining-phlebotomy-chair-with-wheels- 113b&psig=AOvVaw0dq_AYQYKfTrrfWHVe0gaI&ust=1725335816520000&source=images&cd=vf e&opi=89978449&ved=0CBQQjRxqFwoTCICYifyuo4gDFQAAAAAdAAAAABAR  Specimen Collection 2 79 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Equipment Carriers Make blood collection equipment portable. Important in hospital setting and in instances in which patient cannot come to the laboratory Phlebotomy Cart Handheld Carrier https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.medicus-health.com%2Fall-in-one- https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.medicus-health.com%2Fdroplet-phlebotomy-tray-with-small-tube- phlebotomy-cart- rack.html&psig=AOvVaw1lbd7pQpvU2_Sem8FszQeb&ust=1725336132963000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwo tall.html&psig=AOvVaw1kaqZzNftTNxKV5WoawCGv&ust=1725336207772000&source=images&cd=vfe TCIjGlZCwo4gDFQAAAAAdAAAAABAE &opi=89978449&ved=0CBQQjRxqFwoTCMjE7rGwo4gDFQAAAAAdAAAAABAE  Specimen Collection 2 80 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Gloves and Glove Liners standard requirement when performing phlebotomy (CDC & OSHA) new pair must be used for each patient and removed when procedure is completed Nonsterile, nitrile, neoprene, polyethylene, and vinyl examination gloves are acceptable for most phlebotomy procedures a good fit is essential https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.indiamart.com%2 https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.merlin- avoid latex (allergy) Fproddetail%2Fthermo-scientific-nitrile-gloves-labserv- medical.co.uk%2FDisposables%2FGloves%2Fc%2FGloves-279402&psig=AOvVaw11yIpfE5wl-X6s8j9O_J- 25129771691.html&psig=AOvVaw11yIpfE5wl-X6s8j9O_J- J&ust=1725336777801000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNjq4MKyo4gD J&ust=1725336777801000&source=images&cd=vfe&opi=89978449&ved=0CB FQAAAAAdAAAAABAI QQjRxqFwoTCNjq4MKyo4gDFQAAAAAdAAAAABAQ no powder (contaminate tests and also allergy) skin liners are available for persons who develop allergies or dermatitis from wearing gloves barrier hand creams https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.ridgemerino.com%2Fproducts%2Fridge-merino-glove- liner&psig=AOvVaw3GmXko- 5qKjfLprUPTh0GI&ust=1725336893016000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCKiyx_yyo4gDFQAAAAAdAA AAABAE  Specimen Collection 2 81 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Antiseptics Substances used to prevent sepsis. Prevent or inhibit growth and development of microorganisms but do not necessarily kill them. Use in living tissue https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.sylprotec.com%2Fen%2Fantiseptics-topical- solutions%2F3006-benzalkonium-chloride-antiseptic-pads- 70% Isopropyl alcohol https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.fruugo.ae%2F100pc 013100box.html&psig=AOvVaw3xahp2bSVMookiJoj4uZ1s&ust=1725337332824000&source=images&cd=vfe&op sbox-alcohol-swabs-pads-antiseptic-sterilization-alcohol%2Fp- i=89978449&ved=0CBQQjRxqFwoTCJC9zcq0o4gDFQAAAAAdAAAAABAE 74610941&psig=AOvVaw27QpQznIGvGBFAaEEZ_bbi&ust=1725337084063000&s ource=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNiS7tKzo4gDFQA AAAAdAAAAABAE Most common Povidone-Iodine For blood culture and blood gas specimen collection Benzalkonium chloride (Zephiran) Chlorhexidine gluconate https://www.google.com/url?sa=i&url=https%3A%2F%2Fonlinemedicalsupply.com%2Fcollections%2Fcleansers- debriders&psig=AOvVaw1dcr_Hv2CpH2qECHenjje1&ust=1725337126156000&source=images&cd=vfe&opi=899 78449&ved=0CBQQjRxqFwoTCJi_r-mzo4gDFQAAAAAdAAAAABAE https://www.google.com/url?sa=i&url=https%3A%2F%2Fse.pinterest.com%2Fpin%2F50693650178492272 5%2F&psig=AOvVaw0ebPja1BaS1VVHDgoceiX9&ust=1725337413169000&source=images&cd=vfe&opi=89 978449&ved=0CBQQjRxqFwoTCNDj4PC0o4gDFQAAAAAdAAAAABAE  Specimen Collection 2 82 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Needle and Sharps Disposal Containers Red/ Yellow with biohazard label Rigid Puncture-resistant Leakproof Disposable https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.whizz.ae%2Fproduct%2 F3954946%2Fd-h-medical-sharps-disposal-container-3-pack-biohazard-needle- container-1-quart-size-safe-lock-containers-for-disposal-of-syringes-blades-lancets- top-tattoo-supplies-disposal- https://www.google.com/url?sa=i&url=https%3A%2F%2Fcnwtc2.en.made-in- china.com%2Fproduct%2FlxopWPTHaFcY%2FChina-Sharps-Disposal-Container-Biohazard- Medical-Waste-Safety-Box-Plastic-Sharp-Container-for-Tattoo- Needles.html&psig=AOvVaw2Lftg_9IaJRDerZmkSjxxN&ust=1725337641586000&source=images& Have locking lids to seal the kit%2F&psig=AOvVaw2Lftg_9IaJRDerZmkSjxxN&ust=1725337641586000&source=im cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNi99-C1o4gDFQAAAAAdAAAAABAX ages&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNi99- C1o4gDFQAAAAAdAAAAABAn contents when filled to the appropriate volume  Specimen Collection 2 83 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Biohazard Bags leakproof plastic bags that are commonly used to transport blood and other specimens from the collection site to the laboratory with biohazard label have an outside pocket in which requisitions and other forms can be placed. Protects the collector and others from biohazard contamination https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.amazon.ae%2FKEEFITT-Biohazard- Specimen-Bags-100PCS%2Fdp%2FB087CN6ZPR&psig=AOvVaw22iQ5uN- Will contain leak should spill NqCpebSFIwT4gb&ust=1725338073666000&source=images&cd=vfe&opi=89978449&ved=0CBQ QjRxqFwoTCLCQ2Kq3o4gDFQAAAAAdAAAAABAE occur  Specimen Collection 2 84 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Vein-Locating Devices portable illumination devices make it easier to locate veins that are difficult to see or feel. typically shine high intensity LED or infra red lights through the patient’s subcutaneous tissue to highlight veins. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.hopkinsmedicalproducts.com%2Fproduct%2FVenoscope-II- 548803&psig=AOvVaw0xhCAdt4uigHLX33JJHaZK&ust=1725338361546000&source=images&cd=vfe&opi=89978449&ved= 0CBQQjRxqFwoTCLCZrrS4o4gDFQAAAAAdAAAAABAE Hemoglobin absorbs light , causing vein to stand out as dark lines. examples: Venoscope II, Accuvein (AV 300) https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.accuvein.com%2Fassets%2FAV0056 7_E-AV300-Data-Sheet_Print.pdf&psig=AOvVaw141aQgrWfOG1cNB- hbCPhb&ust=1725338450583000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoT CODCl9-4o4gDFQAAAAAdAAAAABAE  Specimen Collection 2 85 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Tourniquet applied or tied around a patient’s arm prior to venipuncture to compress the veins and restrict blood flow. distends or inflates the vein, making them larger and easier to find. also stretches the vein walls so https://www.google.com/url?sa=i&url=https%3A%2F%2Fm.indiamart.com%2Fproddeta https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.trinitysterile.com%2Fproducts%2Fph they are thinner and easier to il%2Ftourniquet-cuff-colour- lebotomy-tourniquet-r-b-1-x-18-cs- 12829767430.html&psig=AOvVaw1FuoRwPu4_0o69bbMcNlEf&ust=17253387947490 1200&psig=AOvVaw1FuoRwPu4_0o69bbMcNlEf&ust=1725338794749000&source=images&cd=vf 00&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNjorIS6o4gDFQA e&opi=89978449&ved=0CBQQjRxqFwoTCNjorIS6o4gDFQAAAAAdAAAAABAK AAAAdAAAAABAQ pierce with a needle. Should be applied in less than 2 minutes disposable  Specimen Collection 2 86 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE Needles Sterile Disposable Single-use Types Hypodermic Multi-sample Winged infusion (butterfly) Gauge: indicated by a number that is related to the diameter of the https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.mls.be%2Fen%2Fp%2Fblood- collection%2Fvacuette-preanalytics%2Fvacuette-accessories%2Fblood-collection- lumen. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.advance https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.nipro- needles%2Fmulti-sample-needles- suppliesuk.com%2FProducts%2FMedical_Products%2Fiv- group.com%2Fen%2Four-offer%2Fproducts-services%2Fstandard-hypodermic- sterile%2F&psig=AOvVaw1N492WNTF_2JjuEjymGaQY&ust=1725339190742000&source=images& products%2F20837&psig=AOvVaw0kG4o_1aeeiRqvRQc9vIaR&ust=17 needle&psig=AOvVaw3INfNkYnGC2bTnDnkL0SbX&ust=1725339140880000&source=i cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCOCBvMO7o4gDFQAAAAAdAAAAABAE 25339252728000&source=images&cd=vfe&opi=89978449&ved=0CB mages&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNCJmqm7o4gDFQAAAAAdA A needles diameter and QQjRxqFwoTCJjQpuC7o4gDFQAAAAAdAAAAABAY AAAABAE gauge have an inverse (opposite) relationship, that is, the higher the gauge number, the smaller the actual diameter of the needle.  Specimen Collection 2 87 EQUIPMENT & MATERIALS USED IN VENIPUNCTURE WINGED INFUSION SET Butterfly (For Syringe Technique) Luer Lock (For Evacuated Tube Technique) Safety Multifly Needle (For Monovette Technique) https://www.google.com/url?sa=i&url=https%3A%2F%2Fm.indiamart.c https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.sarstedt.com%2Fen%2Fproducts%2 om%2Fproddetail%2Ftop-winged-infusion-set- https://www.google.com/url?sa=i&url=https%3A%2F%2Fcavalascientific.com%2Fprod Fdiagnostic%2Fvenous-blood%2Fneedles- 16679632755.html&psig=AOvVaw3txH45KehdqPzjyztffOZs&ust=1725 uct%2Fbd-butterfly-winged-needles- adapters%2Fproduct%2F85.1640.235%2F&psig=AOvVaw3vTlcZn0GJ2cibGszpLi8e&ust=1725339 339339288000&source=images&cd=vfe&opi=89978449&ved=0CBQQ 21g%2F&psig=AOvVaw3txH45KehdqPzjyztffOZs&ust=1725339339288000&source=i 506153000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCIjlndW8o4gDFQAAA jRxqFwoTCPCLiaG8o4gDFQAAAAAdAAAAABAJ mages&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCPCLiaG8o4gDFQAAAAAdAAA AAdAAAAABAE AABAZ  Specimen Collection 2 88 SITE / LOCATION OF VENIPUNCTURE https://www.google.com/url?sa=i&url=https%3A%2F%2Femedicine.medscape.com%2Farticle%2F1998221- overview&psig=AOvVaw35UNfeaHsjgWjctXFmPgea&ust=1725341831073000&source=images&cd=vfe&opi=8997 8449&ved=0CBQQjRxqFwoTCJjMmavFo4gDFQAAAAAdAAAAABAJ  Specimen Collection 2 89 TYPES OF VEINS Large prominent veins A trainee’s dream Be careful not to insert the needle too far. Run the needle along the vein, never through it.  Specimen Collection 2 90 TYPES OF VEINS Large deep veins Be guided by what you can feel rather than what you can see. This skill is described as palpation of the veins. Good veins may lie just below the surface of the skin & can only be located by touch. Take your time locating the exact position of the vein until you are confident of where to insert the needle.  Specimen Collection 2 91 TYPES OF VEINS Small thready veins May be more difficult to deal with. Use a finer bore needle. If the blood is drawn out too quickly, the veins flatten & become useless.  Specimen Collection 2 92 TYPES OF VEINS Superficial veins Tiny veins on the surface of the arms. Not of a suitable size for blood- taking https://www.google.com/url?sa=i&url=https%3A%2F%2Femedicine.medscape.com%2Farticle%2F1998221- overview&psig=AOvVaw2RQQEqttMwowESeeKSIdEG&ust=1725351571284000&source=images&cd=vfe&opi=89978449&ved =0CBQQjRxqFwoTCJjxpNDpo4gDFQAAAAAdAAAAABAJ  Specimen Collection 2 93 TYPES OF VEINS Floating veins May move when you try to insert the needle. You will need to ‘anchor’ the vein about 3-5 cm below the puncture site, with the thumb or forefinger.  Specimen Collection 2 94 TYPES OF VEINS Thrombosed veins These are veins that have been ‘overused’ & are hard & lumpy to the touch.  Specimen Collection 2 95 TYPES OF VEINS Very deep veins Often found in very obese patients. Use palpation to try to find a vein. Ask for assistance if you are unable to find a suitable vein.  Specimen Collection 2 96 STEPS IN VENIPUNCTURE  Specimen Collection 2 97 Step 1: Receive and review test request Test request is prepared and made by the attending physician It can be in written or electronic form Contains identification details of the patient, test(s) requested by the physician  Specimen Collection 2 98 Step 2 : Approach, Identify, and Prepare the Patient Verify patient’s identity Explain the purpose of the procedure Assure the patient  Specimen Collection 2 99 Step 3: Verify Diet Restrictions & Latex Sensitivity  Specimen Collection 2 100 Step 4: Sanitize hands and put on Gloves  Specimen Collection 2 101 Step 5: Position Patient, Apply Torniquet, and ask patient to make fist  Specimen Collection 2 102 Step 6: Select Vein, Release Torniquet, and Ask Patient to Open fist  Specimen Collection 2 103 Step 7: Clean and air-dry the site  Specimen Collection 2 104 Step 8: Prepare the equipment  Specimen Collection 2 105 Step 9: Re-apply Tourniquet and Uncap Needle  Specimen Collection 2 106 Step 10: Ask Patient to make a fist, anchor vein, and Insert Needle  Specimen Collection 2 107 Step 11 : Establish blood flow, release tourniquet, and ask patient to open fist  Specimen Collection 2 108 Step 12 : Fill, remore, and Mix tubes in Order of Draw/ Fill Syringe  Specimen Collection 2 109 Step 11 : Place dry cotton on top of needle, remove needle, activate safety feature of the needle, and apply feature  Specimen Collection 3 110 POST VENIPUNCTURE CONCERNS PRE & POST-VENIPUNCTURE REMINDERS Examine depth, width, direction & type of veins→ Take care not to pass the needle through the vein. Insert the needle at an angle of 30⁰ into the selected vein using a sliding not stabbing motion. Never remove the needle before releasing the tourniquet→ lead to bleeding into the tissues & hematoma→ A swelling that contains blood. Never dispense blood through the needle→ cause hemolysis of the sample. If bleeding hasn’t stopped. Apply yourself further firm pressure Problems encountered in Phlebotomy 1. Always ask someone for help if: You cannot feel or see a suitable vein. You have made two unsuccessful attempts (maximum) to obtain a sample. You do not feel confident of obtaining a sample. 112 Problems encountered in Phlebotomy 2. The vein starts to swell during the venipuncture. Withdraw needle & apply very firm pressure to the puncture site. Warn the patient that he/she will probably develop a bruise at the puncture site 113 Problems encountered in Phlebotomy 3. Blood stops flowing into the syringe after a successful venipuncture. The needle might have slipped out of the vein. The needle might have gone through the vein. Note: It may be possible to move the position of needle slightly to restart the blood flow. However, if this causes pain or distress to the patient it is advisable to withdraw the needle & take the sample from another site. 114 Problems encountered in Phlebotomy 4. Fainting (Syncope) Before fainting, patient turns pale, clam or vague. Always talk to patient during blood-taking to assure all is well. If patient seems going to faint, loosen any tight clothing at the neck or waist. If patient is sat down, put his head between his knees or lower than his heart level. Stand front of him in case he loses consciousness. Shout for help→ will need assistance to support fainting patient. If possible, lie patient down, put a pillow beneath his head & another raising his feet. When he feels able, encourage a sitting position for a while before attempts to stand. When patient feels better, return slowly to upright position & offer a drink. Never allow a patient to leave after a faint until you are confident that he is well enough to go. 115  Specimen Collection 3 116 CAPILLARY BLOOD COLLECTION (SKIN PUNCTURE) Capillary Blood Collection Sites The great finger or ring finger of adults. The medial & lateral plantar surface of the feet of the new born. SITES FOR CAPILLARY PUNCTURE Equipment and Materials 1. Gloves 2. Lancet, safety lancet or automatic lancing apparatus. 3. Sterile swabs. 4. Dry gauze pads or cotton wool balls. 5. Blood collection tubes “capillary tubes” 6. Adhesive dressings 7. Surface disinfectant Procedure 1. Put on the surgical gloves. 2. Check all patient details → as seen in venipuncture. 3. If patient is an adult, briefly explain the procedure. 4. If patient is a child, spend a little time getting to know him or her to give reassurance. Small children should be seated on a parent’s lap Older children may wish to sit on a chair with the parent beside them. 5. Ask parent to hold the child’s free hand. Examine the fingers of the other hand. If the hand feels cold, immerse it in warm water for a few minutes. This will improve circulation & allow blood to flow more freely. Procedure 6. Clean puncture site with an alcohol swab & allow to dry. 7. Remove lancet from its protective covering & hold it firmly between the thumb & forefinger of one hand. Hold the patient’s finger firmly between the thumb & forefinger of your other hand. 8. Press the lancet firmly into the finger. 9. Wipe away the first drop of blood → contains excess tissue fluid & give inaccurate results. 10. Collect blood, drop by drop, into blood container. Apply gentle pressure to make each drop appear. Do not squeeze the finger too hard → causes dilution of sample with tissue fluid or cause hemolysis. Procedure 11. If you are collecting blood into tube with powdered anticoagulant, make sure blood is mixed in the tube ASAP→ gently knock drops down inside tube by tapping the base on to hard surface after every few drops. 12. When enough blood has been collected, place sterile gauze on to the puncture site. Apply pressure to puncture site for a few minutes to stop bleeding. 13. Apply a dressing. 14. Label all specimen tubes. 15. Clean work area & wash your hands with soap & water. HEMATOPOIESIS HML 2013_CLINICAL HEMATOLOGY 1 Monday, September 30, 2024  Hematopoiesis 124 HEMATOPIESIS Also referred to as HEMOPOIESIS It is the continuous, regulated process of renewal, proliferation, differentiation, and maturation of blood cell lines. Results in the formation, development, and specialisation of all functional blood cells that are released from the bone marrow into the circulation. https://www.google.com/url?sa=i&url=https%3A%2F%2Foncohemakey.com%2Fhematopoiesis%2F&psig=AOvVaw2lXmyF5oC2yInTsteKo5hB&ust=1 725688103630000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCPCXsabPrYgDFQAAAAAdAAAAABAQ  Hematopoiesis 125 SITES OF HEMATOPOIESIS In fetus 0 – 2 months (yolk sac) 2 – 7 months (liver & spleen) (until 2 weeks after birth) 5 – 9 months (BM) In infants BM→ (Medullary Hemopoiesis) All bones In adults BM of central skeleton & proximal ends of long bones Vertebra, sternum, ribs, skull, sacrum, pelvis & proximal ends of femur & humorous) (Medullary Hematopoiesis).  Hematopoiesis 126 SITES OF HEMATOPOIESIS Infancy blood cells are produced within all the bone marrow Childhood during the first 4 years of life, nearly all the marrow cavities contain red active marrow with very few fat cells. Then, there is gradual replacement of active marrow by fatty tissue throughout the long bones. Adult Active marrow confined to central skeleton: skull, sternum, ribs, vertebrae, pelvis & proximal ends of femur & humeruS NOTE: Fatty marrow may change back to red active marrow if necessary  Hematopoiesis 127 PHASES OF HEMATOPOIESIS MESOBLASTIC Yolk sac HEPATIC Liver Spleen MYELOID Red Bone Marrow  Hematopoiesis 128 MESOBLASTIC PHASE Place: Yolk sac (blood islands) Main products: Primitive erythroblasts Hemoglobin (Hb): Gower I & II, Portland.  Hematopoiesis 129 HEPATIC PHASE Period: Starts (4-5 wks of gestation) Ends: 1-2 wks after birth. Place: Primary: liver ((2-7) months of gestation). Secondary: spleen Products: Definitive hematopoiesis (RBC’s, WBC’s). Hemoglobin: Hb-F https://www.google.com/url?sa=i&url=https%3A%2F%2Fanatomytool.org%2Fcontent%2Fleiden-drawing-arteries-pancreas-duodenum-liver-and- spleen&psig=AOvVaw3lI0f9BJMDSWBxpjl62EkQ&ust=1725693843165000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNCBz9nkrYgDFQAAAAAdAAAAABA E  Hematopoiesis 130 MEDULLARY PHASE Period: Starts: 5th month of gestation. Continues throughout the life. Place: B.M (only site for hematopoiesis after 2rd wk of birth). Products: Various stages of maturation can be seen in all three lineages Hemoglobin: Hb-A Hb-A2 https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.cancer.gov%2Fpublications%2Fdictionaries%2Fcancer-terms%2Fdef%2Fbone- marrow&psig=AOvVaw1CyMLKvgoiC5ndWPymnh68&ust=1725694103994000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCNic3 NPlrYgDFQAAAAAdAAAAABAE Hb-F  Hematopoiesis 131 EXTRAMEDULLARY HEMATOPOIESIS Liver & spleen may produce blood cells in adults if necessary. Blood cells production outside the bone marrow (B.M) (i.e., liver & spleen) in adults. Indications: To increase blood cells production (exceeding B.M capacity) So, expansion of hematopoiesis occurs down the long bones & even extramedullary.  Hematopoiesis 132 HEMATOPOIETIC HIERARCHY CD34+, CD38- Self-renewal Differentiates into various cell lines. Like small/medium sized lymphocyte (appearance). Give rise to descendents of restricted development to a specific line. Morphologically unrecognized 1st recognizable cell in lineage Mitotically active Functionally active Mitotically inactive  Hematopoiesis 133 ANATOMY OF THE RED BONE MARROW Bone marrow forms a suitable environment for stem cells to survive, grow & develop. It is composed of stromal cells, microvascular network. Stromal cells include: Adipocytes (Fat cells) Fibroblasts Endothelial cells & macrophages Osteoclasts & osteoblasts Reticulum cells Red Bone Marrow: Hematopoietic cells Yellow Bone Marrow: Fat / Adipose tissue https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.anatomyatlases.org%2FMicroscopicAnatomy%2FSection04%2FPlate0456.shtml&psig=A OvVaw0Jdqobp6fcfwh- 6DgnFh5d&ust=1725737457495000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCPjWvZWHr4gDFQAAAAAdAAAAABAR  Hematopoiesis 134 REGULATION OF HEMATOPOIESIS There should be a balance between blood cell production (Hematopoiesis) and normal cell death (Apoptosis) except at the times of requirement. Apoptosis: Programmed cell death Hematopoiesis is regulated by Growth Factors/Hormones)  Hematopoiesis 135 HEMATOPOIETIC GROWTH FACTORS Hormone like substances, also called cytokines & interleukins (IL). Glycoproteins present in minute amounts (picograms) Usually affect more than one lineage Usually act on stem, progenitor cells & on mature cells Usually show synergistic or additive interactions with other growth factors Biological effects of GFs are mediated by specific receptors on target cells  Hematopoiesis 136 HEMATOPOIETIC GROWTH FACTORS  Hematopoiesis 137 GENERAL MORPHOLOGIC CHANGES DURING CELL MATURATION Decreased Cell diameter Decreased nuclear diameter Loss of nucleoli Condensation of Nuclear chromatin Decreased basophilia in the cytoplasm Loss of Nucleus (Erythrocytes)  Hematopoiesis 138 ERYTHROPOIESIS (Erythrocytes) GRANULOPOIESIS (Granulocytic Leucocytes) HEMATOPOIESIS MEGAKARYOPOIESIS (Thrombocytes) LYMPHOPOIESIS (Lymphocytes) ERYTHROPOIESIS HML 2013_CLINICAL HEMATOLOGY 1 Monday, September 30, 2024  ERYTHROPOIESIS 140 ERYTHROPOIESIS It’s a regulated process of RBCs production Erythropoiesis occurs in the BM Erythron: It’s the entire mass of mature & immature RBC’s in the body There is a balance between RBC’s production & destruction. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.shutterstock.com%2Fsearch%2Feryt hropoiesis&psig=AOvVaw2mSpAceExn5EfSW0JuQx49&ust=1725741251495000&source=images& cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCPiBz6aVr4gDFQAAAAAdAAAAABAR  ERYTHROPOIESIS 141 PROCESS OF ERYTHROPOIESIS Proliferation: The division of immature cells to produce increased numbers of more mature cells. Maturation The development of cells through early, intermediate & late stages.  ERYTHROPOIESIS 142 THREE POINTS OF CONTROL OF ERYTHROPOIESIS Control of the self-replication of progenitor cells Control the movement of mature cells from bone marrow into blood A feedback control on cell production.  ERYTHROPOIESIS 143 STAGES IN THE DEVELOPMENT OF ERYTHROCYTES  ERYTHROPOIESIS 144 PROERYTHROBLAST Earliest recognizable erythroid cell in the bone marrow Size (12 - 20 µm) Round nucleus centrally placed within the cell with visible nucleoli & chromatin strands that are dispersed Cytoplasm is usually only a narrow rim around the large nucleus, deep blue (RNA) when stained by Romanowsky dyes  ERYTHROPOIESIS 145 EARLY ERYTHROBLAST/EARLY NORMOBLAST/BASOPHILIC NORMOBLAST Slightly smaller than Pronormoblast Size: 10 - 16 µm in diameter. Nucleus: occupies less of the cell & does not contain nucleoli with more condense chromatin Cytoplasm: predominant color is blue (RNA) with little pinkish color This is a sign that Hb, which stains pink, is being produced & the RNA & protein synthetic apparatus is being lost.  ERYTHROPOIESIS 146 POLYCHROMATIC NORMOBLAST / INTERMEDIATE NORMOBLAST Smaller than basophilic normoblast but with twice amount of Hb Cell size: 8 - 14 µm in diameter. Nucleus is smaller & denser. Nuclear chromatin is condensed Cytoplasm shows a polychromatic staining reaction. Pinkish color predominate This means that it takes both the acid & basic stains producing a purple-pink color. Normally not seen in PB  ERYTHROPOIESIS 147 ORTHOCHROMATIC NORMOBLAST/NUCLEATED RBC Size: has a diameter of 8 - 10 mm Nucleus is eccentric, small, solid & blue black with clumped chromatin Cytoplasm predominantly pinkish (Hb) Mitochondria are no longer evident Normally not seen in PB  ERYTHROPOIESIS 148 POLYCHROMATOPHILIC ERYTHROCYTE / RETICULOCYTE A young RBC that still contains some fine basophilic material→ bluish stain (RNA remnant) Size: 8 - 10 µm in diameter Nucleus is extruded Retics is released to blood, there it needs (1-2) days before maturation Normally, rarely found in PB Retics can be stained with a supravital stain as brilliant cresyl blue or new methylene blue, by which reminant RNA is stained blue & appear as filaments or granules  ERYTHROPOIESIS 149 MATURE ERYTHROCYTES Biconcave disc with circular outline (7 - 8) µm in diameter. (1.5 - 2.5) µm in thickness Stained pink (Hb) Paler central area (2-3) μm No nucleus or mitochondria Life span: 120 days  ERYTHROPOIESIS 150 MATURE ERYTHROCYTES Each pronormoblast divides into 14 – 16 mature RBCs RBCs production takes about 10 days from the pluripotent stem cell to the mature RBCs. From reticulocyte to fully mature RBCs, approximately 1 - 2 days.  ERYTHROPOIESIS 151 REGULATION OF ERYTHROPOIESIS Erythropoiesis is regulated by the hormone erythropoietin (EPO)→ by increasing number of progenitor cells committed to erythropoiesis. The human erythropoietin gene is located on chromosome 7. EPO is produced by (90% kidney & 10% by the liver) Oxygen level in kidney’s tissues controls EPO production. When O2 supply to renal tissue falls, EPO levels increase When O2 supply to renal tissues increases, EPO levels falls  ERYTHROPOIESIS 152 OTHER FACTORS NECESSARY FOR ERYTHROPOIESIS It must be remembered that although erythropoietin plays a critical role in RBCs production, other factors are also necessary for the formation of fully functioning cells. The main ones are listed below: 1. Metals: iron & cobalt 2. Vitamins: Vitamin B12,folic acid & vitamin (B6 pyridoxine) 3. Hormones: Stem Cell Factor (SCF), IL-3 & androgens 4. Amino acids: for protein production  ERYTHROPOIESIS 153 RETICULOENDOTHELIAL SYSTEM (R.E.S.) R.E.S. consists of the spleen, liver & bone marrow. RE cells “macrophages” destroy RBCs by process of phagocytosis. This mechanism is called “extravascular Destruction” of the red cell. On average, 1% of the body’s RBCs are destroyed & replaced each day. This means that 2.5 million cells are replaced each day by bone marrow.  ERYTHROPOIESIS 154 INEFFECTIVE ERYTHOPOIESIS Normally: Some of immature RBC’s don’t develop normally in BM (1- 15%). So, they don’t reach maturity & circulation. They are removed by macrophages in BM. In ineffective erythropoiesis: Number of these cells (not developing into mature RBC’s) abnormally increase ˃ 15%. ERYHTROPOIESIS HML 2013: CLINICAL HEMATOLOGY  Topic Outline Definition of Erythropoiesis Development of Erythrocytes Staining of cells Stages of Erythropoiesis Regulation of Erythropoiesis Ineffective Erythropoiesis  Definition of Erythropoiesis It’s a regulated process of RBCs production Erythropoiesis occurs in the BM Erythron: It’s the entire mass of mature & immature RBC’s in the body There is a balance between RBC’s production & destruction.  Definition of Erythropoiesis Erythropoiesis is the process of RBCs production & includes the following: 1. Proliferation: The division of immature cells to produce increased numbers of more mature cells. 2. Maturation The development of cells through early, intermediate & late stages. 3. Control of Hemopoiesis This occurs at three points during erythropoiesis: a. Control of the self-replication of progenitor cells b. Control the movement of mature cells from bone marrow into blood c. A feedback control on cell production.  Development of erythrocytes: Reduce cell volume/ size Reduce nuclei volume Condensation of chromatin Loss of nucleoli Reduce RNA in the cytoplasm (blue color) Gradual increase in synthesis of Hb (pink color) Reduction in mitochondria  Staining of cells Principle of staining: To understand the specific features of each stage of the cellular differentiation Blood film is prepared & stained with Romanowsky stain (Giemsa, Wright & Leishman). This stain is a combination of Eosin & Methylene blue..  Staining of cells Depending on the cellular components chemical properties: 1) Eosin (acidic, anionic dye): ✓ Stains cationic (basic) components with red to orange color ✓ Granules of eosinophils & Hb of RBCs 2) Methylene blue (basic, cationic dye): ✓ Stains anionic (acidic) components with blue violet color ✓ Nucleus: DNA, RNA & granules of basophils  Stages of Erythropoiesis Six stages: 1. Pronormoblast. 2. Basophilic Normoblast. (early) 3. Polychromatic Normoblast. (intermediate) 4. Orthochromic Normoblast. “NRBCs” (Late) 5. Polychromatophilic Erythrocyte (Reticulocyte) 6. Mature Erythrocyte 1. Pronormoblast“Proerythroblast” ❑Earliest recognizable erythroid cell in the bone marrow ❑Size (12 - 20 µm) ❑Round nucleus centrally placed within the cell with visible nucleoli & chromatin strands that are dispersed ❑Cytoplasm is usually only a narrow rim around the large nucleus, deep blue (RNA) when stained by Romanowsky dyes 2. Basophilic Normoblast “Early Normoblast” Slightly smaller than Pronormoblast Size: 10 - 16 µm in diameter. Nucleus: occupies less of the cell & does not contain nucleoli with more condense chromatin Cytoplasm: predominant color is blue (RNA) with little pinkish color This is a sign that Hb, which stains pink, is being produced & the RNA & protein synthetic apparatus is being lost. 3. Polychromatic Normoblast “Intermediate Normoblast” Smaller than basophilic normoblast but with twice amount of Hb Cell size: 8 - 14 µm in diameter. Nucleus is smaller & denser. Nuclear chromatin is condensed Cytoplasm shows a polychromatic staining reaction. Pinkish color predominate This means that it takes both the acid & basic stains producing a purple-pink color. Normally not seen in PB  4. Orthochromatic Normoblast “Late Normoblast” (Nucleated RBCs/ NRBCs) Size: has a diameter of 8 - 10 m Nucleus is eccentric, small, solid & blue black with clumped chromatin Cytoplasm predominantly pinkish (Hb) Mitochondria are no longer evident Normally not seen in PB 5. Polychromatophilic erythrocyte “Reticulocytes” Size: 8 - 10 µm in diameter A young RBC that still contains some fine basophilic material→ bluish stain (RNA remnant) Nucleus is extruded Retics is released to blood, there it needs (1-2) days before maturation Normally, rarely found in PB  polychrom 5. Reticulocytes Retics can be stained with a supravital stain as brilliant cresyl blue or new methylene blue, by which reminant RNA is stained blue & appear as filaments or granules  Reticulocytes count test Assessment of reticulocytes is a r