Dr Ahmad Mohammad Ashshi (AIBMS)Consultant Virologist

Head of Clinical Laboratory Medicine, Hera General HospitalOn Honorary Teaching Panel, Faculty of Medicine, Umm-Al Qura

University, Makkah Head of Laboratories committee, Saudi Council for Health Specialties

Member of Laboratory Team, Central Board of Accreditation for Healthcare Institutions

Head of Quality Team, M.O.H, Makkah .

Uses of laboratory test To provide information relating to a clinical

diagnosis To investigate the etiology of a clinical condition To study complications of a disease or its

treatment To provide prognostic information To monitor natural history or response to Rx. for a Condition To detect subclinical disease (screening) For Research Purposes

Proper procedures must be followed to assure the integrity of the specimen and its safe and expedient delivery to the laboratory as well as its

processing once in the laboratory

It is the job of the phlebotomist to make sure that all the guidelines of the Laboratories are met and implemented in order to deliver a suitable sample for analysis so that an accurate and precise result will be issued

Several essential steps are required for every successful collection

procedure

Identify the patient.Assess the patient's physical disposition (i.e.

diet, exercise, stress, basal state).Check the requisition form for requested

tests, patient information, and any special requirements.

For body fluid collection provide the patient with the suitable container and collection instruction .

Select a suitable site for venipuncturePrepare the equipment, the patient and the

puncture site.

Perform the venipuncture.Collect the sample in the appropriate

container.Recognize complications associated with

the phlebotomy procedure.Assess the need for sample recollection

and/or rejection.Label the collection tubes at the bedside

or drawing area.Promptly send the specimens with the

requisition to the laboratory

WHAT IS THE MOSTIMPORTANT THING YOU

NEED FOR SPECIMENCOLLECTION???

Patient identification

Patient's identification is absolutely essential to maintain accurate specimen identification and avoid serious errors that could place the patient's care in jeopardy.

 The phlebotomist must correctly identify the patient before specimen collection.

Proper patient identification MANDATORY.

The two unique identifier will be

The patient’s full name

Medical record number

Patient’s Preparation Specimens collected during this "fasting"

period will yield the most reliable results.

Patients should be informed that urine and some of the blood specimens are generally collected during the early morning hours after waking and before eating, usually about 8 to 12 hours after the last ingestion of food or drink

Patient’s Preparation,cont.There are some medications and other

substances that can alter the results of the analyses. That’s why, the lab staff should be informed about the kind of medication ( e.g. Factor s Assay)

There are certain tests performed after administration of drugs / substances such as: glucose tolerance tests (GTT) performed after glucose administration or dynamic tests (of stimulation or inhibition) for different hormones ( e.g. provactive test for growth hormone

urine collection Random Urine specimen Random Urine specimen (drug abuse)(drug abuse)First-morning (First-morning (microscopic examination, b-microscopic examination, b-

HCG, HCG, Clean -catch specimen (MSUClean -catch specimen (MSU ) For urine culture24 hours urine For quantitative chemical 24 hours urine For quantitative chemical

determinations in urine determinations in urine (such as creatinine, protein, calcium, magnesium, sodium, potassium,)

Suprapubic specimen Suprapubic specimen especially for infantespecially for infant

Urine collected from children Urine collected from children

collection bags with hypoallergenic skin adhesivecollection bags with hypoallergenic skin adhesiveCatheter specimenCatheter specimen

Patient’s Preparation factors Therapeutic Drug Monitoring: different pharmacologic agents have patterns of

administration, body distribution, metabolism, and elimination that affect the drug concentration as measured in the blood. Many drugs will have "peak" and "trough" levels that vary according to dosage levels and intervals. Check for timing instructions giving by the physician for drawing the appropriate samples.

Stress: May cause transient elevation in white blood cells

(WBC's) and elevated adrenal hormone values (cortisol and catecholamines). Anxiety that results in hyperventilation may cause acid-base imbalances, and increased lactate.

Patient’s Preparation factors ,cont.تأثير تحت العناصر تأثير انخفاض تحت العناصر انخفاض

المجهودالمجهودالعناصر العناصر زيادو تأثير زيادو تأثير تحت تحت

المجهود المجهود

DecreaseDecrease% % testtestIncreaseIncrease% % testtest

44BilirubinBilirubin1111ACPACP

1111IronIron4141GPTGPT

11LDHLDH33GOTGOT

88PotassiuPotassiumm3131ALPALP

1212Total Total lipidlipid11CalciumCalcium

11ChlorideChloride

33CholesterolCholesterol

1717CreatinineCreatinine

1212PhosphoruPhosphoruss

33Total Total ptoteinptotein

33UreaUrea

44Uric acidUric acid

Effects of Exercise: Muscular activity has both transient and longer lasting effects. The creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and platelet count may increase

Postural Change in posture from supine to erect or sitting causes a shift in fluid from the intravascular to the interstitial space of about 12%.

Most cellular and macromolecular analytes are 5 to 15% higher when specimens are collected erect as compared to supine.

Conversely, moving from upright to supine can have a dilutional effect owing to an increase in plasma volume.

Postural effects are accentuated in patients with edema and low plasma protein values.

Patient’s Preparation factors ,cont.

Diurnal variation: Diurnal rhythms are body fluid and analyte fluctuations during the day.

For example : Serum cortisol levels are highest in early morning

but are decreased in the afternoon.• Serum iron levels tend to drop during the day. • Some sex hormones need special timing as regard

cycle :

(1)FSH,LH (3rd day of cycal {follicular phase})(2)Progesterone (21 day of cycle{ luteal phase})

Other Factors: Age, gender, and pregnancy have an influence on laboratory testing. Normal reference ranges are often noted according to age.

Patient’s Preparation factors ,cont

Requisition form:

It should contain the proper information in order to process the specimen. :A requisition form must accompany each sample submitted to the laboratory. This requisition form must be reviewed to understand

What tests are requested? What specimens are to be collected? What containers are required to collect the

specimens? When the specimens are to be collected? What is the amount of specimen necessary for

testing? What is the collection order of tubes?

The essential elements of the requisition : form are

Patient's surname, first name, and middle initial. Patient's Medical record Number . Patient's date of birth, sex and nationality. Requesting physician's complete name, signature

and stamp. Diagnosis. Source of specimen. This information must be

given when requesting microbiology, cytology, fluid analysis, or other testing where analysis and reporting is site specific.

Date and time of collection. Indicating the test(s) requested.

The essential elements of the requisition cont.,

Histopathology specimen request must contain in addition to the above listed information, the following:

name of the procedure, no. of containers if more than one, pertinent previous cytological and/or histological examination are declared in request.

PAP smear requests must contain, in addition to the above listed information, the following:

Date of last menstrual period (LMP)Previous abnormal reportsTreatment and/or biopsy information

EQUIPMENT:

Evacuated Collection Tubes -.

Needles - The gauge number indicates the bore size: the larger the gauge number, the smaller the needle bore. Needles are available for evacuated systems and for use with a syringe, single draw or butterfly system.

Holder/Adapter - use with the evacuated collection system.

Tourniquet - Wipe off with alcohol and replace frequently.

EQUIPMENTcon,.Alcohol Wipes - 70% isopropyl alcohol.Povidone-iodine wipes/swabs - Used if blood

culture is to be drawn.Gauze sponges - for application on the site from

which the needle is withdrawn.Gloves - can be made of latex, rubber, vinyl, etc.;

worn to protect the patient and the phlebotomist.Syringes - may be used in place of the evacuated

collection tube for special circumstances.Adhesive bandages / tape - protects the

venipuncture site after collection

Collection Tube Additives

Heparin EDTA Citrate Clot Activator Serum Separator

Tube/ Container rTube ContentRequired VolumeInvestigationInstruction

Plain tube with no additiveSome contain clot activator

All Serology tests4-7 mlDon't invert

If the container contain clot activator Invert slowly

several times

Indirect combs test (ICT) ,Chemistry ,Horm

ones except ACTH ,3-4 ml

Depend on No. of tests

Lithium HeparinOsmotic fragility test ,

plasma determination in chemistry (eg. glucose ,

GTT,GCT)

3-4 mlInvert slowly several times to ensure mixing and prevent

clot formation

EDTA

CBC,DC, Retics count, sickle cell, G6 PD, Hb Electrophoresis, DCT,Glycosylated Hb A1c,

ACTH, E.S.R

2 – 3 mldepend on the label on

the tube

Invert slowly several times to ensure mixing and prevent

clot formation

HBV DNA,HCV RNA

4 ml

Serum gel separator tube (SST)

The walls of the SST tube are coated with silica

particles, a clot activator. Initial activation occurs when

blood enters the tube and contact the particles on the

tube wallthe gel forms a physical

barrierbetween serum and blood cells during centrifugation

Serology ,chemistry ,hormones

4-7 ml for serology3-4 ml for chemistry

To continue the activation process , it is

necessary to thoroughly mix the

blood and particles by inverting the tube five

times Don’t freeze SST tubes

Must no be used for therapeutic drug levels, the gel may lower values Not for Blood Bank use

.

Colour of StopperTube ContentRequired VolumeInvestigationInstruction

Sodium citrate

For plasma Coagulation studies (eg, PT,PTT ,FDP

D-Dimer and factors assays

(Blood to anticoagulant ratio (9:1))

Nine volumes of blood are added to one volume of anticoagulant

Ensure tube fills correctly according to volume on

labelInvert slowly several times to

ensure mixing and prevent clot formation

Sodium citrateE.S.R

4 volumes of blood are added to one volume of

anticoagulant)4:1(

Ensure the presence of Anticoagulant,

Invert slowly several time to ensure mixing.

HeparinABG1 mlThe syringe should be immersed inside ice

+

Plain tube without AdditiveSome contain clot activator

ABO ,X-MATC5 ml

Don't invertIf the container contain clot

activator Invert slowly several times

EDTA

2 ml Invert slowly several times to ensure mixing and prevent

clot formation

Colour of StopperTube ContentInvestigationRequired VolumeInstruction

ADULT : with resin*aerobic =blue *anaerobic =yellow without resin :*aerobic = grey *anaerobic = golden

Blood culture

3-10 ml

Bottle should not be refrigerated

1-3 ml PEDIATRIC :

Pink

Plain & Non sterile

Urine analysis , Bence Jones Proteinurine Pregnancy Test10-15 ml

Sample delivery should not exceed 1

hour

Protein/creatinin ratioCalcium/creatinin ratio

Stool/A & C/S,Occult bloodBody Fluid CytologyCollected volume

Sample should not be collected from diapers

or tissue paper

Colour of StopperTube ContentInvestigationRequired VolumeInstruction

Plain & sterileWet smear for Trichomonas

Urine cultureSemen culturePus disharge

SputumBronchial aspirate, tracheal aspirate, urethral disharge

,

The collected volume

All body fluids ( CSF Peritoneal,pleural

,synovial(,Not less than 0.5

ml

Sterile

Wound c/sHVS

CervicalEye ,ear , nasal, umbilica, Mouth

Should be with transport media

Swab should not be dry

Un Sterile swab & alkaline peptone water

Rectal swab C/ S for Vibrio cholera

Un Sterile swab & Cary Blair or selenite broth

Rectal swab C/S for salmonella & shigella

slide

Slide w/ covered Petri dish and placed in media

Fungus CultureKOH examination

Corneal scraping C/S

Fixed smearCytology Pap SmearThe smear should be

fixed

Blood should NEVER be poured from one tube to another since the tubes can have different additives or coatings.

Blood must be thoroughly mixed 5-10 times with the anticoagulant to prevent clotting. DO NOT SHAKE THE TUBE.

Serum Separator tubes should also be inverted. There is a clot activator present and inversion expedites the clotting process

Plastic versus Glass Tubes Plastic tubes have replaced glass tubes for

most uses Less breakage, cheaper, lower weight, more

friendly to the environment. Clot activators are needed in plastic tubes Samples in plastic tubes generally give

equivalent results to glass tubes, but there are

differences for some analytes.

EDTA (Anti-Coagulant

K2EDTA is used to collect whole blood for hematology studies and plasma for analytes with heparin interference

Acts by binding calciumNominal concentration of 1.5 mg/mLRecent move to K2EDTA from K3EDTA for

hematology to reduce affect on RBC parameters

EDTA Side-EffectsEDTA is hyperosmolar causing cell shrinkage

but the low pH of EDTA counterbalances this effect because low pH causes cells to expand.

EDTA induced psuedothrombocytopenia caused by in vitro antibody mediated platelet clumping and platelet satellitism that results from changes in membrane structure that occur when calcium is removed by EDTA.

Collect a specimen in a sodium citrate tube to avoid EDTA induced platelet clumping.

Heparin (Anti-Coagulant)Used to collect whole blood or plasmaBinds to anti-thrombin III to inhibit Xa, IXa,

and thrombinNominal concentration of 12 – 30 U/mLHeparin binds calcium so ionized calcium

must be collected using “Calcium Titrated” or

“Electrolyte Balanced” heparin

Citrate (Anti-Coagulant)Citrate is used for collection of coagulation tests Acts by binding calciumNominal concentration of 3.2% (0.109 mol/L)Recent move to 3.2% from 3.8% to get more consistent results for Prothrombin Time,

particularly for more sensitive reagentsTubes must be properly filled to within +/- 10%

of assigned collection volume

Clot Activator Shortens Time to ClotIn vitro activation of clotting system to

enhance clot formationA silica clot activator is attached to the tube

wall with a silicone surfactant Requires mixing for optimal affectTime to clot is 15-30 minutes instead of 1

hour

Serum Separator Gel•Separates Serum from Cells

•Polymer gel with specific gravity between that of

serum and cells

• Migrates and forms a barrier during centrifugation

•Separation of cells from serum stops metabolic and

•hemolytic effects

•Certain analytes and therapeutic drugs may bind to

gel over time

Labelling the Sample

A properly labelled sample is essential so that the results of the test match the patient. The key elements in labelling are:Patient's surname, first and middle.Patient's ID number.NOTE: Both of the above MUST match the

same on the requisition form.Date, time and initials of the phlebotomist must

be on the label of EACH tube

Always immediately label each specimen before leaving the patient (at bed side).

Never: Pre-Label blood specimen tubes before drawing blood.

Never give the specimen to someone else to label.

Verify again that the information on the labeled specimen matches that on the request form.

  Unlabeled specimen(s) or incorrectly

labeled specimen(s)• All blood specimens that are mislabeled or

unlabeled will be redrawn.

• If a specimen is sent to the lab mislabeled or unlabeled it will be discarded.

• All blood, whether unlabelled or mislabeled, intended for cross matching, must be redrawn, without exception

If the specimen is irreplaceable the ordering physician must attest to the correct patient’s identity and must authorize (on the specimen relabeling form) affixation of the correct label by an attendance nurse.

Example of specimens considered to be irreplaceable Obtained from internal body site or obtained as part

of invasive procedure (e.g. Bronchoscopy specimen, bone marrow or CSF).

Critically timed specimens (e.g. prior to antibiotics or dosing schedules).

Neonatal specimens. CORD BLOOD specimens that are received with the mother's information on the label can be relabeled with the baby's labels. A nurse must do the relabeling.

Collection Volume Under filling occurs because:

Tube was removed too quickly.

Tube slips back from vacutainer needle.

Air drawn in from butterfly or connector tubing.

Expired tubes with low vacuum (rare)

Impact of Incorrect Volume on PT and APTT The APTT is the most sensitive to variations

in final citrate concentration. It increases by

an average of 3% for 10% underfilling and 10% for a 20% underfilling.

The PT increases by an average of 5% for 20% underfilling and by 15% for 30% underfilling.

Order of DrawA. Evacuated Tube

Method B. Syringe Method C. Microtainer Collection

Method (skin puncture heel or

fingerstick )

Blood gases

SLIDES/SMEAR

Other additive tubes

ErrorsAnticoagulant cross contaminationCitrate : If drawing a blue top tube for coagulation

tests, (other than PT and PTT), a red tube (2-3 cc) must be drawn first to avoid contamination from tissue thromboplastin, which can yield false coagulation Results.

EDTA : A calcium chelating agent; can yield false magnesium and calcium results if drawn before a red top tube. Can falsely elevate potassium results if drawn before a green top tube.

Oxalate : Interferes with cell membranes

Reasons for a Rejected Specimen1. No Test Requisition Form (TRF) 2. Test not written on TRF 3. Submitted wrong specimen for test

requested 4. Quantity Not Sufficient (QNS)

5.5. Missing or inadequate identiificationMissing or inadequate identiification

6.6. Clotted Clotted

Avoiding Common Errors in Specimen Collection1. Insufficient quantity 2. Hemolysis 3. Failure to use the correct container for

specimen collection 4. Inaccurate and incomplete patient guidance 5. Failure to label a specimen correctly and to

provide all pertinent information 6. Failure to tighten specimen container lids,

resulting in leakage and/or contamination of specimen

ADDITIONAL CONSIDERATIONS

To prevent a hematoma:Puncture only the uppermost wall of the veinMake sure the needle fully penetrates the upper most wall of the vein. (Partial penetration may allow blood to leak into the soft tissue surrounding the vein by way of the needle bevel)

Remove the tourniquet before removing the needle

Use the major superficial veinsApply pressure to the venipuncture siteAvoid a probing, traumatic venipuncture

To prevent hemolysis (which can interfere with many tests):

Mix tubes with anticoagulant additives gently 5-10 times. Avoid drawing blood from a hematoma, IVs and catheters Draw slowly when collecting with syringes Avoid drawing

the plunger back too forcefully, and avoid frothing of the sample.

Make sure the venipuncture site is dry ( allow alcohol to dry before collection).

Avoid a probing, traumatic venipunctureUse a larger bore needle (20 gauge) Draw from antecubital fossa rather than the hand

•

To Avoid Clots

Mix specimen thoroughly after collection.

Blood should be drawn quickly & transferred immediately from syringe to tube.

Do not overfill tubes , add the indicated amount on the tube .

Prolonged Tourniquet ApplicationMetabolic and Concentrating EffectsApplication of a tourniquet for >1 minute can result in

hemoconcentration, causing an increase in the concentration of large molecules (e.g. serum proteins) that are unable to pass through the capillary wall.

Total protein, iron, total lipids and cholesterol increase from 5%-7%, bilirubin increases 8% and AST 9%.

Prolonged tourniquet use also promotes anaerobic glycolysis resulting in an increase in plasma lactate, decreased pH, and an increase in blood potassium.

SAFETY AND INFECTION CONTROL

PROTECT YOURSELFPractice universal precautions:

Wear gloves and a lab coat or gown when handling blood/body fluids.

Change gloves after each patient or when contaminated.

Wash hands frequently.Dispose of items in appropriate containers.

Dispose of needles immediately upon removal from the patient's vein. Do not bend, break, recap, or resheath needles to avoid accidental needle puncture or splashing of contents.

Clean up any blood spills with a disinfectant such as freshly made 10% bleach.

If you stick yourself with a contaminated needle: Remove your gloves and dispose of them properly. Wash the area well with soap and water. Record the patient's name and ID number. Follow institution's guidelines regarding treatment and

follow-up.

PROTECT THE PATIENT

Place blood collection equipment away from patients, especially children and psychiatric patients.

Practice hygiene for the patient's protection. When wearing gloves, change them between each patient and wash your hands frequently. Always wear a clean lab coat or gown.

Specimen Transport Specimens should be delivered to the laboratory

promptly after collection in biohazard bag

Rapid transport samples eg: PTH & glucose If delay is inevitable it is generally better to refrigerate samples.However refrigeration may itself cause artefactual changes in the results.

Some Samples need ice and anaerobic condition while transportation eg: Arterial blood gases and ammonia

    normal bacteria will multiply producing contaminated normal bacteria will multiply producing contaminated samplesample

If the organism urase producer, ammonia release If the organism urase producer, ammonia release will increase Ph resulting in destruction of cells will increase Ph resulting in destruction of cells and castand cast the bacteria will break down any glucosethe bacteria will break down any glucose

RBC, WBC, Cast will lyzeRBC, WBC, Cast will lyze

Protein conc will alterProtein conc will alter

Bilirubin and Urobilinogen oxidized-not detectedBilirubin and Urobilinogen oxidized-not detected

Uric acid and urate deposited to for oxalate or Uric acid and urate deposited to for oxalate or phosphate crystalphosphate crystal

If the urine sample left at RT

Comments and Questions