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Sample collection and processing Part 1

Dr. Urender singh2nd yr PG Biochemistry

Faculty of Medicine & Health ScincesSGT Medical College

Gurgoan

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• Accuracy -To ensure that analytical results obtained are representative of actual analyte conc. in patient and, thus of his/her physiological/pathological state.

• Safety• Minimize patient discomfort and

complications• Avoid recollection

Aim of sample collection

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• Blood- venous/ arterial/ capillary- whole blood ; serum ; plasma• Urine• Faeces• Saliva• Solid tissues• Amniotic fluid• Cerebrospinal fluid• Synovial fluid• Peritoneal fluid• Pleural fluid• Pericardial fluid

Example of biological specimen

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Venipuncture

Skin puncture

Arterial puncture

Blood collection

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Personal protective equipments(PPE)1. lab coat2. Gloves3. mask4. Eye protection

Proper hand hygiene now a days alcohol-based antiseptic hand cleaners are used instead of hand washing if the

hands are not visibly soiled if not available and clean visibly soiled hands are there,detergent should be used followed

by alcohol based cleaner isolation this separate certain pts from others and limit their contact with hospital

personnel and visitor Handle all blood specimens as potentially infectious material. Never recap needles ,dispose in puncture

proof sharps container

Standard precautions for infection control

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1. Phlebotomy Room- private area such as an isolated room• counter or work table for all of the blood handling equipment and

supplies• phlebotomy chair should be available 2. Blood Processing Room-• refrigerated centrifuge• a small refrigerator • sufficient counter space for the processing of blood specimens.• A -70°C freezer should be in or near the room• sink and running water .• Use one counter area for processing the blood samples and another

counter area for completing paperwork.

Facilities required

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Disposable latex gloves (use non-latex, e.g. nitrile or vinyl, if employee and/or patient has a latex allergy).

Isopropyl alcohol Gauze or lint free tissues Needle with safety feature Venous blood collection system (e.g,Vacutainer)

Syringe (if vacuum blood collection system unavailable) Safety transfer device (if using syringe) Tourniquet (preferably latex free) or blood pressure cuff cont……

Equipment Preparation

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Puncture proof container marked"Bio hazardous".Red and yellow biomedical waste disposal bagsBand Aids (optional)Blood specimen collection tubes appropriate for tests orderedDisinfectant (10% household bleach) for bench topsOther personal protective equipment, e.g., lab coat or face shield, if appropriate

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1.Patient Preparation 1. Identification— at least 3 items of identification should be used• Name• Age• Sex• Medical record / C R No.• Room no. ,bed no.• Address for outpatient

For alcohol levels testing and medicolegal cases –chain of custody must be established . Additional identification like photograph may be required.

2. Enquire for allergy to latex , alcohol , band aid any

uses

Venipuncture

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Timed specimens –

a. to monitor changes in a patient's condition—eg. monitor a cardiac marker to determine if it is rising or decreasing

b. to measure how well a substance is metabolized –eg. Two hour postprandial specimen and/or a glucose tolerance test4. position of patient- patient should be comfortably seated or supine ,and should have been in this position for at least 20 mins before the specimen is taken

3. Timing of blood collection Specific instructions must be followed a.Circadian variation b.Travel c.Menstrual calendar

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5. Verify –If a fasting specimen is required, confirm that the fasting order has been followed- To be fasting the patient should not have consumed even a lifesaver, stick of gum or other sugar-containing food items during the fasting period, or has “accidentally” ingested a sip of juice, black coffee, tea, single bite of other food items, or any amount of alcohol.h/o of any long distance travelh/o of exercise/ stenous physical activity within 8 hoursh/o of smoking in past one hourh/o of alcohol ingestion h/o any intake of medicines ,herbal preparationsh/o of any bleeding disorder h/o of fainting on blood collection such factors should be mentioned on the requisition slip of the patient

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The vein sites should be examined and considered in the following order:

• Median Cubital • Cephalic • Basilic (avoid if possible because it is

closest to the brachial artery and median nerve)

• Wrist Area (top and side only) • Back of the Hand • Lower Extremity/Foot/Ankle avoid

performing on diabetic patients or those with obvious circulatory problems)

• Scalp (performed only by highly experienced phlebotomists)

Site of venipuncture

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Preparation of Venipuncture Site -

Using sterile alcohol prep. pad (70% isopropyl alcohol), cleanse site using concentric outward moving circles. Allow site to air dry. Do not blow on site.

Using index finger, palpate (feel) for vein, its direction and location

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Apply the Tourniquet 3-4 inches above venipuncture site 60mmHg pressure if blood pressure cuff is used

Precautions when using a tourniquet: Do not apply tourniquet above a central venous catheter or midline, on arm with a kidney access device (Cannula, Fistula, Shunt).

The tourniquet should not be left on the site for longer than one minute during the actual collection due to the possible occurrence of hemoconcentration and infiltration of blood into tissue. If the patient is sensitive to the tourniquet, apply over sleeve or towel.

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• Changes in serum composition when venous occlusion is prolonged from 1 to 3 mins.

• Increased-total protein,Fe,total lipid, cholesterol,billirubin• Decreased-potassium.

• Composition of blood drawn first, is most representative of Composition of circulating blood.

• Hence first drawn tubes should be for analytes like calcium ,that are importent to critical medical decisions.

• Conc. Of protein bound analytes is also increased due to stasis.

Effects of prolonged venoocclusion

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Never leave the tourniquet on longer than one minute during the examination. If more time is needed, release the tourniquet and then re-apply.

If a vein is not readily apparent, the following techniques may be used to make palpation easier:

• Massage arm or apply warm cloth to dilate vein. • Bend arm very slightly, relaxing the muscles of the antecubital. • Lower arm below the heart to allow veins to fill to capacity. • Close patient’s hand to dilate vein. Avoid having the patient

"pump" the hand It falsely elevates potassium, phosphate,

lactate ,lowers pH and hence increases ionized calcium conc.

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Perform the Venipuncture –• vacutainers • Syringes • butterfly needle

Choose appropriate Needle/ butterfly size-Adults -19 to 22Children- 21 to 23

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Hold dry gauze over puncture site with arm raised for 30-60 seconds or until bleeding stops .

If puncture site continues to bleed slightly, apply a pressure bandage to site using clean folded gauze, band-aid or paper tape. Tell patient to leave the bandage on site for at least 15 minutes.

For patients with a known bleeding disorder (Haemophilia, von Willebrand's Disease ), immediately after removing butterfly needle apply direct pressure to puncture site until bleeding stops for up to 10 minutes, even if blood was not obtained.

Label the Specimen Containers Proper disposal of gauze,needle and holder, gloves etc.

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• If swelling is noticed during venipuncture, immediately release tourniquet, remove needle and apply pressure using gauze pad

• On patients with a bleeding disorder (Haemophilia or von Willebrand's Disease), use a 21 No. gauge butterfly needle or smaller. Do not probe if blood is not obtained immediately. Remove needle.

• If your first attempt fails, pull needle back slowly, relocate vein, pull skin taut (anchor vein), and redirect needle. Lateral needle relocation should never be attempted to access the basilic vein, since nerves and the brachial artery are in close proximity.

• Do not attempt to stick patient more than two times; contact another phlebotomist.

• If patient feels a shooting, electric-like pain, tingling, or numbness, remove the needle, notify nurse or physician, and document the incident.

Precautions

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Inappropriate sites for venipuncture

Burn, Scar, Tattoos

Damaged vein

Edema

Hematoma

Arm with cannulas, fistulas or vascular grafts

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Procedure related

• Hematoma formation• Iatrogenic anemia• Inadvertent arterial puncture• Infection of the site• Nerve injury

Patient related

‣ Fainting(syncope)‣ Excessive bleeding‣ Allergies‣ Obesity‣ seizures

Complications

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• method of choice in paediatric patients, especially infants• Venipuncture of deep veins in paediatric patients may also rarely

cause - cardiac arrest - Haemorrhage - thrombosis - venous constriction followed by gangrene - damage to organs or tissues accidentally punctured - infection• Accessible veins in sick infants must be reserved exclusively for

parenteral therapy.

Skin puncture

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Skin puncture is useful in adults with 1) Extreme obesity 2) Severe burns or bandages 3) Extensive vein damage 4)Thrombotic tendencies 5)Sample applied directly to point of care testing or filter paper

Skin puncture is often preferred in old age patients because skin is thinner and less elastic; thus a hematoma is more likely to occur from a venipuncture.

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Adults : Finger tip EarlobeChildren: Heel Big toe (not in <1 year)

Finger stick:

• It may be necessary to warm the skin puncture site to increase blood flow to the site. A warm, moist towel (or other warming devices) at a temperature no higher than 42oC may be used to cover the site for three to five minutes. This technique increases blood flow to the site up to 7 fold, does not burn the skin, and does not result in significant changes for routinely tested analytes.

• Thoroughly cleanse the chosen site with 70% isopropanol. Allow the skin to air-dry.

Sites-

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Obtain the sample from the third or fourth (middle or ring) finger Choose a site that is on the side of the fingertip, midway between the edge and midpoint of the fingertip. The puncture should be made perpendicular to the fingerprint ridgesUse a lancet to make a deep puncture (1.5 mm) at the chosen site Using a dry gauze, wipe away the first drop of blood, making certain the area is completely dry. The first drop is most likely to contain excess tissue fluid.

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Collect the blood sample in a collection device appropriate for the test to be performed.

Do not massage or milk to avoid contamination with tissue debrisApply a piece of gauze, (or cotton ball), to the puncture site, using slight pressure until the bleeding has stopped. For older children and adults, offer a band-aid

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• hold the heel with the forefinger at the arch and the thumb proximal to the puncture site at the ankle. If using a lancet the blade should be not longer than 2.4 mm to avoid injury to the calcaneus (heel bone)

• Collect the specimen in microcollection capillary blood tubes or

• On filter paper for neonatal screening or molecular genetics testing

• After blood collection is complete, elevate the infant’s foot above the body and apply pressure using sterile gauze until bleeding has stopped .Do not apply adhesive bandages.

Heel stick

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Skin puncture blood is a mixture of undetermined proportions of blood from arterioles ,venules , capillaries, interstitial and intracellular fluids.

Venous vs capillary serum

Capillary>venous

% Capillary =venous

Capillary <venous

%

Glucose 1.4 phosphate bilurubin 5.0

potassium 0.9 urea calcium 4.6

chloride 1.8

sodium 2.3

Total protien 3.3

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Indications-• assessment of PO2 when pulse oximetry is abnormal • there may be changes to acid/base status as in

respiratory,metabolic,kidney diseases• Symptoms of oxygen,carbon dioxide or pH imbalance

like difficulty breathing,nausea or vomitting• assessment of ventilator efficacy• Head or neck trauma,injuries that affect breathing• Prolonged anesthesia as in cardiac bypass or brain

surgery• Rapid Hb

Arterial puncture

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Site-

Adult -In order of preference, the radial, brachial, and femoral arteries can be selected. Unacceptable sites are those that are irritated, edematous, near a wound, or in an area of an arteriovenous (AV) shunt or fistula

Neonate- indwelling umbilical artery catheter

Children- capillary puncture to obtain arterialized capillary blood

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• 1. Have the patient make a fist and occlude both the ulnar (opposite the thumb side) and radial arteries (closest to the thumb) by compressing with two fingers over each artery

• 2. Have the patient open their fist and observe if the patient's palm has become bleached of blood

• 3. Release the pressure on the ulnar artery (farthest from the thumb) only and note if blood return is present. The palm should become perfused with blood. Adequate perfusion is a positive test indicating that arterial blood may be drawn from the radial artery. Blood should not be taken if the test is negative. Serious consequences may result if this procedure is not followed and may result in the loss of the hand or its function

Modified Allen Test

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• The amount of anticoagulant should 0.05 mL liquid heparin (1000 IU/mL) for each milliliter of blood.

• Using too much heparin is probably the most common pre-analytic error in blood gas measurement

• the blood should fill the syringe fairly quickly.

• The colour of the blood should be bright red, not dark red.

Procedure of art. puncture

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• There should be no air space in the syringe at the completion of collection.

• Roll syringe between your hands 15-20 times to mix the anticoagulant in the syringe.

• Label specimen.

• Transport specimen on crushed ice. The specimen should be cool not frozen, (maintain a temperature of 1–5°C) to minimize leukocyte consumption of oxygen

Important Points to remember

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Complications are unusual but can include:

Local pain

Haematomas

A rare vasovagal episode

Extremely rarely, an expanding aneurysm of the radial artery has been reported after frequent punctures

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• Serum- The watery portion of blood that remains after coagulation has occured; it is obtained after centrifugation.

Historically, serum was the preferred assay material for determining extracellular concentrations of constituents in blood.

• Plasma- the noncelloular component of anticoagulated whole blood, plasma contains clotting factors.

Today, plasma is preferred for many, but not all, laboratory investigations because the constituents in plasma are better reflecting the pathological situation of a patient than in serum and because of better turn around time(TAT)

Serum or Plasma?

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PlasmaAdvantage of plasma Time saving: Plasma

samples can be centrifuged directly after sample collection, unlike serum, in which coagulation is completed after 30 minutes

Higher yield: 15 to 20 % more in volume of plasma than of serum can be isolated from the same volume of blood

Disadvantage of plasma.

Contamination with cations: NH4 +, Li+, Na+, K+.

Assay interference caused by metals complexing with EDTA and citrate.

inhibition of alkaline phosphatase

activity by zinc binding,

inhibition of metal dependent cell activation in function tests,

binding of calcium (ionized) to heparin .

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Plasma cont…• Advantage of plasma Prevention of coagulation-

induced interferences: Coagulation in primary and secondary tubes that were already centrifuged, may block suction needles of the analyzers when serum tubes are used; this is prevented by using anticoagulants.

Disadvantage of plasma Interference by fibrinogen in total

protein measurement and protein electrophoresis.

Inhibition of metabolic or catalytic

reactions by heparin .

Interference in the distribution of ions between the intracellular and extracellular space by EDTA, citrate.

Reduced storage stability for certain analytes

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Serum Advantages of serum Nearly cell-free

Good storage stability for most analytes

Wide range of assays available

Disadvantages of serum

Longer TAT Instrument or test interference

from fibrin, especially with anticoagulation therapy

• Increase in the concentrations of platelet components in serum as compared to plasma (e.g. potassium, phosphate, magnesium, aspartate aminotransferase, lactate dehydrogenase, serotonin, neurone-specific enolase, zinc).

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Plasma >serum % Plasma=serum

% Plasma< serum

%

Calcium 0.9 Bilrubin Albumin 1.3

Chloride 0.2 Cholesterol ALP 1.6

LDH 2.7 creatinine AST 0.9

Total protein 4.0 Bicarbonate 1.8

CK 2.1

Glucose 5.1

Phosphorus 7.0

Potassium 8.4

Sodium 0.1

Urea 0.6

Uric acid 0.2

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Sample collection and processing 2nd part

by Dr. Urender singh2nd yr PG Biochemistry

Faculty of Medicine & Health SciencesSGT Medical College

Gurgoan

• Heparin ( mucoitin polysulfuric acid)-effective anticoagulant in small quantities without significant effect on many determinations.

• Heparin is available as lithium heparin (LiHep) and sodium heparin (NaHep) in green top tubes

• Heparin accelerates the action of antithrombin III, neutralizing thrombin and preventing the formation of fibrin. 2 mg of calcium/sodium/lithium/ammonium salt used /10 ml blood (1000 units /ml)

• lithium heparin may be used for most chemistry tests except for lithium and folic acid levels; for lithium, a royal blue sodium heparin, Na2EDTA, can be used instead. Sodium heparin cannot be used for assays measuring sodium levels but it is recommended for trace elements, leads and toxicology.

Anticoagulants and additives

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Disadvantages of heparin are high cost, temporary action, blue background formation in smears stained with wright’s stain, inhibition of acid phosphatase and polymerase activity

Liquid heparin causes dilution error in various analysts. so, dried (lyophilized) heparin is recommended for critical care testing

Calcium-titrated heparin at a concentration of 40 to 60 IU/ml blood (dry heparinisation) and 8 to 12 IU/ml blood (liquid heparinisation) is recommended for the determination of ionized Calcium.

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Ethylenediamine tetra-acetic acid (EDTA) anticoagulant of choice for haematology, isolation of genomic DNA and qualitative and quantitative virus determination by molecular techniques as it preserves cellular components of blood.

It’s available in lavender top tubes as a liquid or spray-dried in the disodium, di- or tripotassium salt form (K2EDTA in plastic, spray-dried, and K3EDTA in liquid form in glass tubes)

K3EDTA is a liquid and will dilute the sample 1-2%. K∼ 2EDTA is spray-dried on the walls of the tube and will not dilute the sample

• Converts calcium to non ionized form• A conc. of 34-38 g/l is used in ratio 1 part to 9 parts of blood

• For coagulation testing, a light blue top tube containing 3.2% sodium citrate is commonly used because it preserves the labile coagulation factors, and its effect is easily reversible with addition of calcium

• Black top tubes also contain buffered sodium citrate and

generally used for Westergren sedimentation rates. They differ from the light blue top tubes in that the ratio

of blood. Anticoagulant is 9:1 in the light blue top tubes and 4:1 in the black top tubes.

Citrate

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• sodium/ potassium/ lithium/ ammonium oxalates form insoluble complexes with calcium ions

• 1 to 2g/l mostly used• >3g/l causes haemolysis • Cause shrinkage of cells by drawing water except

combined ammonium (3parts) +potassium(2parts) oxalates

• Inhibits ALP, ACP, LDH , AMYLASE

oxalates

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• Used as inhibitor of glycolysis (inhibits glycolysis for 24 hrs at 25degree C) in combination with EDTA/oxalate.

• As preservative, effective at concentration of 2g/l of blood. 3 to 5 times higher conc. required when used alone for anticoagulation.

• Prevents glycolysis by forming an ionic complex with Mg++, thereby inhibiting the Mg++ dependent enzyme, enolase

Sodium fluoride

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• Antiglycolytic agent (inhibits glycolysis for 24 hrs) at conc. Of 2g/l

• No effect on urease ( so it is preferred when a single sample we want to do the urea and sugar)

• Inhibits CK

Sodium iodoacetate

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Stopper color Anticoagulant/additive Specimen type/use Mechanism of action

Red (glass) None Serum/chemistry and serology

N/A

Red (plastic/Hemogard) Clot activator Serum/chemistry and serology

Silica clot activator

Lavender (glass) K3EDTA in liquid form Plasma/hematology Chelates (binds) calcium

Lavender (plastic) K2EDTA/spray-dried Plasma/hematology Chelates (binds) calcium

Pink Spray-dried K2EDTA Plasma/blood bank Chelates (binds) calcium

White EDTA and gel Plasma/molecular diagnostics

Chelates (binds) calcium

Light blue Sodium citrate Plasma/coagulation Chelates (binds) calcium

Light blue Thrombin and soybean trypsin inhibitor

Plasma/coagulation Good for fibrin degradation products

Black Sodium citrate Plasma/sed rates – hematology

Chelates (binds) calcium

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Light green/black Lithium heparin and gel Plasma/chemistry Inhibits thrombin formation

Green Sodium heparin, lithium heparin Plasma/chemistry Inhibits thrombin formation

Royal blue Sodium heparin, Na2EDTA Plasma/chemistry/toxicology

Heparin inhibits thrombin formation Na2EDTA binds calcium

Gray Sodium fluoride and lithium iodoacetate

Plasma/glucose testing Inhibits glycolysis

Yellow Sterile containing sodium polyanetholesulfonate (SPS)

Serum/microbiology culture Aids in bacterial recovery by inhibiting complement, phagocytes and certain antibiotics

Yellow Acid citrate dextrose (ACD) Serum/blood bank, HLA phenotyping and paternity testing

RBC preservative

Tan (glass) Sodium heparin Plasma/lead testing Inhibits thrombin formation

Tan (plastic) K2EDTA Plasma/lead testing Chelates (binds) calcium

Yellow/gray and orange Thrombin Serum/chemistry Clot activator

Red/gray and gold Clot activator separation gel Serum/chemistry Silica clot activator

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•During centrifugation, blood is forced into a thixotropic inert, polymer gel (specific gravity 1.04) material located at the base of the tube.

• The gel undergoes a temporary change in viscosity during centrifugation and lodges between the packed cells and the top serum layer .

Serum separator gel

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Special considerations• Stoppers may contain zinc ,interfering with its estimation• Stoppers may contain TEBP ( tris [2 butoxyethyl ] phosphate ) a constituent of rubber which may interfere with measurement of certain drugs.• With time the vacuum in evacuated tubes is lost , this may lead to short draw and incorrect ratio of blood and anticoagulant• The silicone coating also decays with age• Soda lime glass tubes may release trace elements like calcium and magnesium

•Plastic (polyethylene terphthalate) tubes are preferred over glass tubes as they decrease likelihood of breakage and hence exposure to infectious material.

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• Additive carry-over –• This can occur when blood in a additive tube

touches the needle during venipuncture or during transfer from a syringe

• Most problem are seen with EDTA• Least with HEPARIN, as it occurs in blood naturally• Carry-over least likely to occur if tubes fill from the

bottom up which keeps the tube contents away from the needle.

Additives-Compounds added to biological specimen to prevent them from clotting or to prevent the constituents of a specimen.

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This is a special sequence of tube collection that reduces the risk of specimen contamination and additive carry-over.

EXCEPTION: Venous pH and ionized calcium are to be drawn before any tubes, including routine or fungal/AFB blood cultures

Order of draw

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• Designed to deliver blood first for those tests most affected by micro clot formation.

• This method assumes that the blood entering the syringe last is the freshest and that this blood will be the first blood out of the syringe during the transfer process.

• Because the clotting process is activated the minute the blood starts to fill the syringe, it is important to transfer the blood quickly and to fill anticoagulant tubes before the serum tubes.

The order of draw for the syringe system

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order of draw is as follows:1st. Sterile specimens (blood culture tubes or bottles)2nd. Light blue stopper for coagulation studies.3rd. Lavender stopper - EDTA4th. Green stopper - heparin5th. Gray stopper - oxalate/fluoride6th. Red stopper or red/gray stopper (gold Heamogard) - non

additive and gel separator, respectively.7th. Orange stopper - thrombin

The order of draw for the syringe system

The haemolytic sample Hemolysis mean, the libration of hemoglobin after RBC have ruptured. It can occur in-vivo or in vitro. Plasma or serum assume pink to red

colour.

Cause of Hemolysis:-1. Using too small a needle2. Forcing the blood through needle3. Shaking the tube or bulb too vigorously after blood collection4. Presence of excess of anticoagulant 5. Centrifuging blood sample at high speed before completion of

clotting6. Freezing or thawing of blood 7. Presence of water in the container

Chemical Test Affected By Haemolysis

1. S.potassium2. S. inorganic Phosphorus3. SGOT4. SLDH5. S. Acid Phosphatase

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1. Visual detection- possible above 300 mg/LSamples are always intensely red coloured.

2. Spectrophotometric detection For slightly haemolyzed sample, however, appropriate

sample blank and biochromatic reading can be used to eliminate effect of hemoglobin interference.

Detection and measurement of haemoglobin in serum or plasma

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3. Analytical measurement

In vivo vs in vitro hemolysis• haptoglobin is reduced during intra-vessel haemolytic

process• After in-vitro haemolysis all constituents of erythrocytes,

including potassium concentration, lactate dehydrogenase and aspartate aminotransferase activities, increase in addition to the concentration of free haemoglobin in plasma or serum.

• In contrast, haptoglobin concentration in plasma/serum of haemolytic samples remains unchanged.

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• Rise of intracellular constituents in the extra-cellular space Cell constituents with an intracellular concentration 10 times

higher than the extra-cellular concentration will increase in plasma/serum during haemolysis (e.g. potassium, magnesium, phosphate, lactate dehydrogenase, aspartate aminotransferase, aldolase etc )

• Interference with analytical procedure Adenylate kinase released from erythrocytes causes an

increase of creatine kinase and CK-MB activity especially when inhibitors of adenylate kinase in the assay mixture are inadequate

‣ Optical interference by haemoglobin the colour of haemoglobin increases the absorption at a

respective wavelength or changes the blank value.

Mechanisms of interference by haemolysis

The icteric sample

• Icteric plasma contains high levels of bilirubin. Normal levels of bilirubin are about 0.5 mg/dL. In cases of hyperbilirubinemia, levels will exceed 1.5 mg/dL and plasma will become affected.

• Icteric plasma samples have a high prevalence in samples from patients in the ICU, as well as gastroenterology, surgical and pediatric patients. Conc. of bilirubin greater than 2.5 mg/dL can lead to clinically relevant changes of anti-thrombin. Higher conc. can interfere with other coagulation tests.

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• Spectral interference 1. Bilirubin has a high absorbance between 340 nm and

500 nm wavelengths

2. The reduction of absorption as a result of oxidation bilirubin in alkaline solution is the main cause for bilirubin interference in modifications of the Jaffé method without deproteinisation

3. In a strongly acid solution the absorption of conjugated bilirubin shifts to the UV wavelengths. Therefore bilirubin interferes in the determination of phosphate using the phosphomolybdate method through its reducing effect

Mechanisms of bilirubin interference

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• Chemical interference –

1. Bilirubin interferes in oxidase/peroxidase based test systems. Bilirubin reacts with H2O2 formed in the test system which causes systematically lower results in enzymatic procedures that are used for the measurement of glucose, cholesterol, triglycerides, urea and creatinine

2. Bilirubin competitively interferes with dyes binding to albumin

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• The visual inspection of plasma or serum samples for the detection of hyperbilirubinaemia is often not sensitive enough. This is particularly true when samples are simultaneously stained by other pigments (e.g. haemoglobin and its derivatives).

• Hyperbilirubinaemia is directly detected in diluted samples that are measured at 450 and 575 nm . The direct procedure of bilirubin measurement is only applied for the determination of hyperbilirubinaemia in newborns

Detection and documentation of increased bilirubin concentrations in clinical samples

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• With the nutritional supply of carotines or carotinoids, bilirubin concentration by direct measurement is overestimated

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• Method selection -The high prevalence of hyperbilirubinaemia in patients from intensive care, gastroenterological surgical or paediatric departments makes it pertinent to select analytical methods that are less susceptible towards bilirubin interference.

• Blanking procedures are useful to eliminate spectral bilirubin interferences

Prevention of bilirubin interference

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• A mixture of non-ionic tensides may reduce bilirubin interference like in the spectrophotometric determination of inorganic phosphate using phosphomolybdate

• Immunological procedures for the measurement of serum albumin can be used to replace dye binding methods that are susceptible to bilirubin interference

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Lipaemia is a turbidity of serum or plasma which is caused by elevated lipoprotein concentrations and which is visible by the eye.

• Post-centrifugal coagulation of serum samples of heparinized patients can also be the cause of turbidity.

The Lipaemic Sample

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• Increased triglyceride concentration in plasma/serum. This can be due to-

I. Food intake ( after intestinal absorption triglycerides are present in plasma for 6 to 12 h.)

II. Altered lipid metabolism

III. Metabolic disorders causing hypertriglyceridemia

IV. lipid infusions

Causes of lipaemia (turbidity)

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Identification and quantification of lipaemia

Optical and photometric methods for serum and plasma samples

If triglyceride concentrations above 300 mg/dL Lipaemia in plasma or serum is visually observed

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• Patient should fast at least 12 hours before blood samples are taken

• In patients receiving parenteral infusion of lipids a period of 8 hours of interruption of the treatment is necessary

• Centrifugation for chylomicrons – at 1000g for s.lipids – at 12000g for 10 mins• Ultra-centrifugation must be employed for the

separation of low density lipoproteins and high density lipoproteins.

Means to avoid lipaemia and interferences caused by turbidity

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• Optical clearing systems- Commercial test kits may contain detergents such as triton X-100, cholic and desoxycholic acid, lipase or cholesterol esterase to remove turbidity in plasma or serum samples.

The method of choice for removal of turbidity from serum and plasma is a 10 min centrifugation in a micro-centrifuge with 10 000 g.

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Sample collection and processing 3rd part

Dr. Urender singh2nd yr PG Biochemistry

F MHSSGT Medical College

Gurgaon

Handling of specimen for Analysis

1. Identification2. Preservation3. Separation and storage4. Transport

1.Identification Min. information- Name, location, Identifying number,

Date and time of collection. No specific labeling” handling with special

care” All specimens should be treated as special

and potentially infectious.

2.Preservation• For these tests, must be 4 °C from start to until

the specimens are analyzed, e.g ammonia, blood gas determinations, blood pH , acid phosphatase, lactate, pyruvate, and certain hormone tests gastrin and renin.

• Transfer of these specimens to the laboratory must be done by placing the specimen container in ice water.

• A notable decrease in pyruvate and increase in lactate con. within a few minutes at ambient temperature

• Specimens for bilirubin or carotene and for some drugs, such as methotrexate, must be protected from both daylight and fluorescent light to prevent photodegradation.

• For molecular diagnostic laboratories, challenge to recover RNA from transported specimens.

• Specimens from liver, spleen, or heart have large amounts of RNA, but specimens from skin, muscle, and bone have lower RNA content.

• Tissue samples should be frozen immediately. Alternatively, a blood specimen should never be frozen before separation of the cellular elements because of hemolysis and released heme that may interfere.

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• PlasmaCentrifuge the anticoagulated blood for at least 15

minutes at 2000 to 3000 g to obtain cell-free plasma.• SerumWhen coagulation is complete, centrifuge the sample for

at least 10 minutes at a minimum speed of 1500 g.

• When separating serum or plasma, the temperature of lab should not drop below 15 °C or exceed 24 °C.

3.Separation and storage

Plasma/ serum should be separated within 2 hours. Premature separation of serum may permit formation of

fibrin, which can clog sampling devices in testing equipment. If it is impossible to centrifuge a within 2 hours, the

specimen should be held at room temperature rather than at 4 °C to decrease hemolysis.

Plasma should be removed from the primary tube promptly after centrifugation and held at −20 °C .

Frost-free freezers should be avoided because they have a wide temperature swing during the freeze-thaw cycle.

4 °C or −20 °C is not the optimum storage temperature for all tests; some lactate dehydrogenase isoenzymes are more stable at room temperature than at 4 °C.

Specimen tubes should be centrifuged with stoppers for reduce evaporation maintains anaerobic conditions ( carbon dioxide and ionized calcium ) and prevent aerosolization of infectious particles.

Removal of the stopper before centrifugation allows loss of CO2 and increase pH (acid phosphatase )

Cryopreservation- one method to store and maintain samples for extended periods of time of white blood cells and DNA . Whole blood specimens can be centrifuged, and white cells removed and cryopreserved at −20 °C until these cells are required for DNA extraction.

For longer periods of storage, isolated DNA can be stored at-70°C.

After these extracted DNA samples have completely thawed, it is important to fully mix the sample to ensure a homogeneous specimen.

The extracted DNA should not be exposed to repetitive cycles of freezing and thawing because this can lead to shearing of the DNA

Blood Alcohol • Alcohol distributes into the aqueous compartments of

blood; because the water content of serum is greater than that of whole blood, higher alcohol concentrations are obtained with serum as compared with whole blood e.g 1:18

• So serum is the best choice of specimen.• Specimens should be kept capped to avoid evaporative loss.• Blood may be stored, when properly sealed, for 14 days at

room temperature or at 4 °C without preservative.• For longer storage or for nonsterile postmortem specimens,

sodium fluoride should be used as a preservative.

Estimation of Alcohol consumed• Dr. Erik M.P.Widmark, a Swedish physician, N =W• ρ •[Ct + β • t]/(d •Z) N = number of drinks W = body weight (kg) ρ (rho) = volume of distribution (L/kg) (0.68 for males, 0.55 for females) Ct = blood alcohol concentration (kg/L) β = rate of ethanol elimination (0.15 g/L/h) t = time since first drink (h) d = specific gravity of alcohol (0.8) Z = amount of ethanol alcohol per drink (L) (15 mL of ethanol in a

standard drink)• Frequently the time since the first drink is unknown; the formula can

be modified. N =W• ρ •[Ct ]/(d •Z)

4.Transport

• We assumed that transport time is as 72 hours.

• The specimen tube should be good constructed.

• Reduced pressure of 0.50 atmosphere (50 kPa) may be encountered during air transport.

• Polypropylene and polyethylene containers are usually

suitable for specimen transport. Glass should be avoided.

• Not use Polystyrene tube because it may crack when frozen.

• Sample delivery for molecular analysis ( usually DNA only) will be delayed due to extracted nucleic acid, can be transported in a buffer solution or water, or it can be dried down and shipped as a loose powder.

• DNA should be transported at ambient temperatures and should not be exposed to extremely high temperatures because it will begin to degrade.

• Corrugated, fiberboard, or Styrofoam boxes designed to fit around a single specimen tube are commonly used. A padded shipping envelope provides adequate protection for shipping single specimens.

• When specimens are shipped as drops of blood on filter paper (e.g., for neonatal screening), the paper should be enclosed in a paper envelope to ensure that the sample remains dry.

• For transport of frozen or refrigerated specimens, a Styrofoam container should be used. The container walls should be 1 inch (2.5 cm) thick to provide effective insulation.

• The container should be vented to prevent

buildup of carbon dioxide under pressure and a possible explosion. Solid carbon dioxide (dry ice) is the most convenient refrigerant material for keeping specimens frozen, and temperatures as low as −70 °C can be achieved.

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Other biological samples

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• Random –for spot glucose, ketone bodies ,molecular testing for infectious agents such as Chlamydia(STD)

• First-morning sample- most conc. and has a lower pH due to decreased respiration during sleep – used for microscopic examination , for proteins and chorionic Gonodotropin measurement.

• Midstream specimen-best for investigating bladder disorders

Urine

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Timed specimen- obtained at:-

1. specific times of day (urobilinogen 2-4 PM ) 2. during certain phases of micturition e.g. first 10 ml for urethritis , midstream for bladder diseases 3.In some metabolic disorders, urine must be collected during or immediately after symptoms of the disease appear- Acute Neurovisceral Attack in Porphyria is measurement of urinary PBG( porphobilinogen) and Lead exposure increases urinary ALA ( alfa linolenic acid).

24-hour total volume collection-

1 .The preferred container should be 4-L , plastic, and clean with the correct preservative added.

2. Instruct the patient to start with an empty bladder and to end with an empty bladder

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Creatinine excretion is based on muscle mass, and since a patient's muscle mass is relatively constant, creatinine excretion is also reasonably constant. Therefore, one can measure creatinine on several 24-hour collections to assess the completeness of the specimen and keep this as part of the patient's record.

Catheter specimen – urine examination for critically ill and urinary tract obstruction.

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IMPORTENT

Urine should not be collected at the same time for two or more tests requiring different preservatives. Aliquots for an analysis such as a microscopic examination should not be removed while a 24-hour collection is in process.

Removal of aliquots is not permissible even when the volume removed is measured and corrected, because the specific gravity, volume, and composition of the urine varies throughout the day, and test results will be affected.

Collection of Urine from Children

• For Infant scrotal or perineal area is cleaned and dried first, and skin oils are removed. A plastic U-Bag is placed around the infant’s genital area.

• To obtain a sterile urine specimen for culture from an

infant, a suprapubic tap is performed.

Urine Ethanol•Urine has been used as an alternative, less invasive specimen for the determination of alcohol use.

•During the postabsorptive phase following alcohol ingestion, the concentration of alcohol in urine is roughly 1.3 times that in blood.

•However, the use of urine alcohol measurements to estimate blood concentrations is discouraged because the ratio of 1.3 is highly variable.

•The detection of alcohol in urine represents ingestion of alcohol within the previous 8 to 12 hours.

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Result Reason

Change in color Breakdown or alteration of chromogen or other urine constituent (e.g. hemoglobin, melanin, homogentisic acid, porphyrins)

Changes in odor Bacterial growth, decomposition

Increased turbidity Increased bacteria, crystal formation, precipitation of amorphous material

Falsely low pH Glucose converted to acids and alcohols by bacteria

Falsely elevated pH Breakdown of urea by bacteria forming ammonia

False negative glucose Utilization by bacteria (glycolysis)

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False negative ketone Volatilization of acetone. Breakdown of acetoacetate by bacteria

False negative bilirubin Destroyed by light. Oxidation to biliverdin

False negative urobilinogen Destroyed by light

False positive nitrite Nitrite produced by bacteria after specimen is voided

False negative nitrite Nitrite converts to nitrogen and evaporates

Increased bacteriuria Bacteria multiply in specimen before analysis

Disintegration of cells/casts Unstable environment, especially in alkaline urine, hypotonic urine or both

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Preservative for 24 hr sample Tests

None (refrigerate) Amino acids, amylase, chloride, copper, creatinine, glucose, heavy metals (arsenic, lead, mercury), histamine, immunoelectrophoresis, lysozyme, methylmalonic acid, microalbumin, mucopolysaccharides, porphobilinogen, porphyrins, potassium, protein, protein electrophoresis, sodium, urea, uric acid, xylose tolerance

10 g boric acid Aldosterone, cortisol

10 mL 6N HCL Calcium, catecholamines, citrate, cystine, homovanilic acid, hydroxyproline, magnesium, metanephrines, oxalate, phosphorus,

0.5 g sodium fluoride Glucose

Equal amounts of 50% alcohol, Saccomanno's fixative, SurePath or Preserve CT

Cytologic examination

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• For occult blood- “the presence of a bleeding ulcer or malignant disease in the gastrointestinal tract.

• In the newborn, the first specimen from the bowel (meconium) may be used for detection of maternal drug use during the pregnancy .

• Screening for tryptic activity (cystic fibrosis in children)• For analysis of Polio-melliteus virus.• Fecal nitrogen and fat in 72 hour specimen in malabsorption.• Differential and quantitative analysis of stool DNA integrity

proposed as biomarker for detection of colorectal cancer• No preservatives required.• Transport specimen in cold ice box.

Faeces

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Obtained usually by lumbar puncture.USE Diagnosis of meningitis, CVA, metastasis In molecular diagnosis for rapid identification of infectious

agent and T and B cell gene rearrangement in hematological malignancies.

PROCESSING Rapid processing is a clinical requirement. So

antiglycolytic agents usually not added.

CEREBROSPINAL FLUID

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• Prenatal diagnosis of congenital disorders- fetal maturity, Rh isoimmunization, intrauterine infection, cystic fibrosis, sickle cell anemia, Tay-Sachs disease, and thalassemia.

• 10 ml aspirated in syringe• Sterile containers, such as polypropylene test

tubes or urine cups, are used to transport the fluid to the laboratory.

Amniotic fluid

Synovial Fluid Synovial fluid is a clear thixotropic fluid that serves

as a lubricant in a joint, tendon sheath, or bursa. Sterile plain tubes- culture ,glucose and protein

measurements. EDTA tube- total leukocyte, differential, and

erythrocyte counts. Microscopic slides are prepared for staining with

Gram’s or other stains indicated, and for visual inspection.

Pleural, Pericardial, and Ascitic Fluids• The fluid may be removed to determine whether it

is an effusion or an exudate—a distinction made possible by protein or enzyme analysis.

• The fluid may also be examined for cellular elements.

• In the molecular diagnostic laboratory identify the infectious agent.

• Possibly for the detection of cancer cells.

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1. Measurement of blood group substance 2. Determination of secretor status

3. Saliva can be used as a source of DNA or RNA.

4. Ethanol measurements and for the detection of drugs of abuse, but it is not a frequently used sample for ethanol determinations

Saliva

Buccal Cells

• Collection of buccal cells has been identified as providing an excellent source of genomic DNA

• useful after blood transfusions and bone marrow transplantation.

• collect buccal cells: rinsing with mouthwash and using swabs or cytobrushes.

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Malignant tissue for estrogen , progesterone receptors 0.5 – 1 gm

Frozen quickly, within 20 minutes – liquid nitrogen/ dry ice +alcohol

Solid tissue

Breath Ethanol• The fundamental principle for use of breath

analysis is that alcohol in capillary alveolar blood rapidly equilibrates with alveolar air in a ratio of approximately 2100 : 1 (blood : breath).

• Before breath alcohol analysis, a deprivation

period of 15 minutes is required to allow for clearance of any residual alcohol that may have been present in the mouth e.g. very recent drinking, use of alcohol-containing mouthwash, vomiting of alcohol-rich gastric fluid.

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Thank you!