Blood Specimen Collection

Blood Specimen Collection

• Chapter 1 outlines the necessary precautions for sample collection, venipuncture procedure, vacutainer tube identification, and differences between plasma and serum

Types of Specimens

• Common specimen types include blood, sputum, urine, and stool

Test Requisition Essentials

• Requires Patient ID, tests requested, time/date of collection, sample source, clinical data and physician contact information are recorded

Collection Requirements Checklist

• Includes patient preparation/identification, needed sample/container types, proper labeling, special handling, and safety precautions

General Collection Precautions

• Treat all patient specimens as infectious
• Do not recap needles
• Include correctly filled request form and ensure specimens are well labeled
• Match information on container to order form
• Protective wear is required where splashes are possible
• Use puncture-proof, biohazard containers for sharps disposal
• Frequent hand washing and glove use are mandatory in between patients during the phlebotomy procedure

Components of a Phlebotomy Tray

• Request form, syringes/needles, tourniquet, 70% isopropyl alcohol swabs, sterile gauze/adhesive dressings, specimen containers, self-sealing plastic bags, rack, sharps waste container

Phlebotomy Key Points

• Minimize trauma and ensure visible veins for phlebotomy and "strive to give your best first time, every time"
• Avoid infection sites, superficial/small veins, bruised/scarred areas, limbs with IV infusions, areas near phlebitis or edema, previous venipuncture sites, limbs with injury
• Do not use thrombosed, fibrosed, sclerosed, or inflamed veins

Venipuncture/Syringe Assembly Guidelines

• Verify patient identity
• Phlebotomy tray has all the required items
• Do not pre-labeling bottles in advance to avoid errors.
• Reassure patients should be comfortable
• Venipuncture isn't really painless
• Immobilization of limbs for children
• Gloves are required, changed between patients
• Vacuum tube > needle/syringe
• Stoppers not to be removed, pierce rubber stopper and let vacuum draw blood

Proper Sampling Steps

• Firstly, the skin is cleaned with 70% ethanol and air dried.
• A tourniquet applied above the site is only released and reapplied when blood flows into the syringe
• Duration of tourniquet application should be kept to under one minute
• Slowly draw piston of the syringe, never withdrawing blood faster than the vein is filling.
• The use of a minimal tourniquet, an appropriately sized needle, blood being withdrawn slowly, the blood being delivered gently into the tube, and frothing being avoided during mixing with anticoagulant avoid haemolysis.
• After obtaining the blood, remove the needle, then press a sterile swab over the puncture site.
• The swab should be elevated with gentle pressure for a minute on the arm, when checking that bleeding has completely ceased
• Finally, put an adhesive small dressing on the puncture point

Tourniquet Use

• Know how to use before approaching patient
• Apply it 10 cm above the puncture location (3 finger breadths)
• Guarantee patient is comfortable

Capillary Blood Collection

• Reserved for point-of-care tests and when venous access isn't an option (e.g., infants, obesity)
• Performed using a lancet
• In adults/older children, blood is taken from the palmar surface of the 3rd or 4th finger's distal digit (~3-5 mm lateral to the nail bed)

Capillary Blood Collection for Infants

• Use the plantar surface(bottom) of the heel with a sterile disposable lancet
• Preferred site: the heel's lateral side (little toe side)
• Puncture depth on medial side should be controlled.
• The insertion of the lancet should have > 2mm as this can injure nerves
• Vacutainer EDTA tubes collect blood for CBC
• Na3 Citrate tubes collect blood for PT

Vacutainer Tubes

• For PT assays, add nine parts blood to one part sodium citrate
• For ESR assays, add four parts blood and one part sodium citrate
• Plain vacutainer tubes collect blood for chemistry tests

Plasma vs Serum Specimens

• Plasma: contains anticoagulant, cellular components collect at the bottom, liquid portion is straw-colored
• Serum: no anticoagulant, cellular components form clot at the bottom, liquid portion is clear/straw colored

Proper Labeling Protocol

• Identification: ask name, compare, and validate
• Label with: patient name, unique ID, test, and time/date

Manual Cell Counts Indications

• Necessary for CBC parameters exceed instrument linearity, instrument malfunctions, or in resource-limited situations
• Involves hemacytometer, calibrated pipettes, and diluents
• Same principle for WBCs, RBCs, and platelets; varies only in dilution, fluid, and counting area

Hemacytometer Use

• Involves counting chambers, Levy chamber with improved Neubauer ruling being most common
• Each of four corner squares has WBC subdivides into 16, the center square subdivides into 25 for platelets/RBCs

White Blood Cell (WBC) Count

• WBC/leukocyte count is WBC number per liter/microliter of blood
• EDTA-anticoagulated or skin puncture blood dilutes with 2% acetic acid which lysis RBC
• Standard WBC count dilution: 1:20
• Charge hemacytometer with mix and cells are counted in the four corner squares with microscope.

WBC Count Procedure

• Clean hemacytometer/coverslip with alcohol and use lint-free tissue.
• Dilution: 10µL mixed blood with 190µL WBC diluting fluid in a test tube to make 1:20 dilution
• Mix by inversion
• Sit for 10 minutes so red blood cells lyse, occurring within 3 hours of dilution
• Charge both hemacytometer sides and put in a moist chamber for 10 min. to allow the cells to settle before counting
• Place horizontally on microscope, lower condenser, focus using 10x lens so cells evenly distribute.
• Count in four corner squares for 1:20 dilution, using top-left square as start.
• Touching top/left lines are counting cells, not those touching to bottom/right
• Repeat count on other side; difference <=10% for even distribution
• Average WBCs counted on each side
• WBC count Calculation: (W1+W2+W3+W4) x 50 = .../µl

Platelet Count Details

• Platelets are difficult to count platelets because they adhere to foreign objects and smaller in size
• Dilute whole blood with EDTA 1:20 by 1% ammonium oxalate
• Count 25 small squares in hemacytometer center

Platelet Procedure

• 1. Mix 10 μL mixed blood with 190 μL of 1% ammonium oxalate test tube dilution and charge the chamber.
• 2. The hemacytometer charged for 15 mins in a humid chamber
• 3. Count with 40x objective lens in 25 small squares to sides of the chamber
• Equation to calculate: R1+R2+R3+R4+R5 X 1000=… µL

Red Blood Cell (RBC) Count Details

• Rarely done manually because of the inaccuracy; prefer microhematocrit or Hb concentration with automation not available

RBC Procedure

• 10μL blood+1990μL saline/small tube to make a 1:200 dilution and saline used
• Count using 10x objective lens
• The numbers of RBC across the grid should remain close and consistent between slides
• Count x 10000 will derive result

Hemoglobin Concentration Method and Principle

• Cyanmethemoglobin (hemoglobincyanide) is the reference method approved by CLSI
• Involve blood, in an alkaline Drabkin with K ferricyanide,potassium cyanide, bicarbonate, and surfactant
• Conversion of hemoglobin to methemoglobin, then converted to cyanmethemoglobin by K cyanide
• The concentration is proportional to absorbance at 540 nm

Hemoglobin Concentration steps in practice

• Use whole blood anticoagulated with EDTA, heparin, or blood by capillary puncture and 20μL is transfered and mixed to Drabkins then wait about 10 minutes to allow then the solutions is fully converted

• Once conversion if complete transfer to a a cuvette
  

• Set up as blank 
  

• Any standard solution should be ready
  

• Absorbance levels can then determine
  

Hemoglobin Sources of error

• Avoid and store solution in darker shades
• Can cause turbidity which can measure falsely due to high presence of blood or a certain concentration.
• Plasma can be centrifuged to then obtain the top layer/supernatant
• Correct water dilution of cells which cannot be hazed then multiply by number
• Potass. can be used in cases where those are found in patients' globing

Manual Microhematocrit

• The technique involves the volume that RBs occupies in the presence of blood which can then measure the volume
• Both tubes of the volume should have 3/4s with additives and with heparin
• Coloured end dipped with the correct placement; has a layer of 4mm thickness minimum.
• Cent centrifuge the tubes and check blood reading
• And buffy count is not to be included

Sources of errors

• Improper tube sealing causes an error due to leak.
• Decreases hemocrit because and increases if the blood wasn't
• Be more precised with timings to stop rise.
• Cannot read with the buffy

Red cell Indices

• MCV (mean corpuscular volume ) *MCH (mean cell hemoglobin) *MCHC(mean corpuscular hemoglobin) are RCs indices which gives the avg
• MCV range 80-100 fl =normas >100 macro and <80 micro
• MCH the avg weight within a RBC
• MCHC the Hc amount within cell Normor= range of 32-36.

Normal Reference Values for Adults

• Chapter outlines ranges for red blood cell count, Hb concentration, PCV, MCV, MCH, MCHC, white blood cell count, and differential white cell count
• RBC count: Men: 5.0 ± 0.5 x 10^12/l, Women: 4.3 ± 0.5 x 10^12/l
• Hb concentration: Men: 15 ± 2 g/dl, Women: 13.5 ± 1.5 g/dl
• PCV: Men: 0.45 ± 0.05 l/l, Women: 0.41 ± 0.05 l/l
• MCV: Men/women: 92 ± 9 fl
• MCH: Men/women: 29.5 ± 2.5 pg
• MCHC: Men/women: 33 ± 1.5 g/dl
• WBC count: Men/women: 4.0-10.0 x 10^9/l
• Differential white cell count: Neutrophils: 2.0–7.0 x 10^9/l, Lymphocytes: 1.0-3.0 x 10^9/l, Monocytes: 0.2-1.0 x 10^9/l, Eosinophils: 0.02-0.5 x 10^9/l, Basophils: 0.02–0.1 x 10^9/l
• Platelet count: Men/women: 140-440 x 10^9/l

Blood Film Preparation

• Chapter addresses manual/automated methods and Romanowsky dye solution preparation
• Blood film prepared on clean glass slide from fresh, non-anticoagulated or EDTA-anticoagulated blood
• Placing a small drop of blood on the center line, spread to create a thin film using the spreader at a 30 degree angle in steady motion until the blood separates
• Dry in air

Blood Film Preparations Precautions

• The spreader should be dried
• The film should be about 3cm and end 1 cm before point
  • Thickness of film is managed by pressure and speeds angles
  • Use large for the less anemic
• Slide on is microscopy
• After the slide is used after. it shouldn't be overflown

Blood Marrow + Romanosky

• The process should happen in first few hours before drying
• It's important not to until dry.
• Common in Leishman, Wright or Giemsa stain.
• With proper uptake of the primitive cells will cause a positive affect
• While the gran contain the acidic alkaline and heparic affinity
• The routine of the RM helps see distinct stains granules, and staining diffs dependent on the parts

Stain Prep

• 0.2g dye to flask over 2mL methanol and warmer the mixture for about fifteen at 50 degrees

Automated Machine

• Will run each process by itself if has enough
• Some will have a stained horizon
• Be aware of problems regarding the process

Preparation of Bone Marrow Films

• To prep bones first have to be dried then stain them with dyes, can last over an minimum of 2o mins.

Cell Seperation

• Have certain separation methods to help conc.
• Buffy coat will go through a separation layer

Collection Process

• To perform spin and EDTA within 150 g with super plasma careful
• Use a fine pipette
• Deposit the plate on the layer
• Use film stain the usual

Buff coat benfits

• Detect different prim and atypical issues on it
• Can determine fungus, macrophage, cells to name a few
• Is the the blood of those w/pancyt and erythr

Tick Film and Para

_ Thick film via a small drop that takes up an area and is spread

• Film, at minimum, has go be 30mins
• Not to be burned, avoid mess during washes

erythrocyte formation

• Erythrocytes, requires a lot formation, separation, and production in the blood cell flow
• Initial stem cell is the specification that produces development to red b
• Dev is through characterises for degenerating cells which are then called out of cell
• Erythrop is under certain regulations, factors, and presences that help the process

rbc composition

• Stain use
• Hb is overal and can weigh 6800 d, there are molecules for function in lung, adult but all

Reference ILOS

• Must outline the anemias and defects of synthesis in the cell and what it does
• The Hb synth and the overall identification of the issues

Classification

• Mainly focuses on abnormalities that in all categories

Blood count

• Membrane
• Energetic defects

anemia overview

• Where the Hb in the bodt is reduced and isn't within normal range

anemia clinical features

• Signs are easy of fatigue that causes disp on exertion

Causes of anemia

• Deficiency and loss of the factors

morphocla

• Can recognize overally causes by their MCV count
• All different types are included

Iron Investigations

• Serum ferritin correlates with tissue iron stores
• Ferritin increases will damaged tissue
• The method can be the Elisa option

Lab Tech

• In regards to the disorder there the use of folate analys will help the iron

• The serum levels

• Erythrocyte Sedimentation Rate (ESR) measurement: Measurement of the sedimentation of red cells in the blood in hour

• Tube recommendations of: length about 30 cm, diameter no less than 2.5 mm

• Should be cleaned and free of dust