CHAPTER 12 PART TWO (specimen handling and processing)

Possible sources of preanalytical errors before collection

Altitude, dehydrated patient, duplicate test orders, exercise, inadequate fast, incomplete requisition, medications, patient stress, pregnancy, smoking, strenuous exercise, wrong test ordered, treatments (ex- intravenous medications, radioisotopes)

Possible source of preanalytical errors at time of collection

Misidentified patient, Antiseptic not dry, Expired tube, Failure to invert additive tubes properly, Faulty technique, Improper vein selection, Inadequate volume of blood, Inappropriate use of plasma separator tube or serum separator tube, Incorrect collection tube, Incorrect needle position, Incorrect needle size, mislabeled tube, mixing tubes to vigorously, Non-sterile site preparation, Wrong collection time, Patient position, Underfilled tube, Prolonged tourniquet application

Possible source of preanalytical errors during specimen transport

Agitation induced hemolysis, Delay in transporting, Exposure to light, Failure to follow temperature requirements, Transport method (ex- hand carried vs pneumatic tube)

Possible sources of preanalytical errors during specimen processing

Contamination, Delay in processing or testing, Delay in fluid separation from cells, Evaporation, Failure to centrifuge specimen, according to test requirements, Failure to separate fluid from cells, Incomplete centrifugation, mislabeled aliquot, multiple centrifugations, rimming of clots

Storage after testing is part of the

Postanalytical phase but can also be apart of the preanalytical phase when repeat testing or additional tests are ordered on a specimen.

A delay in separating the blood cells from plasma or serum can result in

Metabolic changes in the sample

Rough handling and agitation can

Hemolyze specimens, Activate platelets, And affect coagulation tests, As well as break collection tubes

Tubes should be transported

Vertically with the stopper up to reduce agitation that can cause red cell damage and lead to hemolysis in the specimen

The upright position also allows the blood to drain away from the tube stopper to minimize the chance of aerosol (a fine mist of specimen) release when the stopper is removed during processing or testing.

In addition, the upright position aids clot formation in serum tubes and prevents the clot from sticking to the stopper. Blood in contact with tube stoppers in gel tubes may end up in the serum or plasma above the gel barrier after centrifugation. This can contaminate the specimen with blood cells that can affect test results and fibrin strands or clots that can cause blockages in testing instruments.

General transportation guidelines

Specimens are typically placed in biohazard bags. Bags must be leak proof and have a biohazard symbol on them along with a pouch for paperwork. If the laboratory is on site, specimens are either hand delivered by the phlebotomist or sent to the laboratory by means of an automated transportation system such as a pneumatic tube, vertical track, or robot system

Pneumatic tube system (PTS or P-Tube)

One of the most common means of transporting specimens to the laboratory from other areas of a hospital. This type of delivery system consist of a network of long tubes that connect to sending and receiving stations in certain laboratory areas to stations located in various areas of the hospital, usually near a nurses station

The system works by propelling special canister-like carriers through the system tubes using compressed air or partial vacuum. Must be packaged correctly in a leak resistant container and sealed in zipper type plastic bags fitted with disposable clear plastic liners in case of leakage. Carriers must contain foam pads and special padded liners to provide contaminant and cushioning during transport.

Tests that are not affected by PTS transport

albumin, alkaline phosphatase, AST, chloride, creatinine, glucose, sodium, total bilirubin, total protein, BUN, uric acid, thrombin time, WBC concentration

Tests that are affected by PTS transport

Tests negatively affected by PTS transport are those influenced by red cell damage, these include:

Potassium, plasma hemoglobin, acid phosphatase, and lactate dehydrogenase. Specimens that must be maintained at body temperature, such as cold agglutination and cryoglobulin are also not recommended for PTS transport.

Off site transportation

Many patient specimens are delivered to clinical laboratories from offsite locations, such as physicians offices, Clinics, Patient service centers, Or private homes

Local courier or mobile phlebotomist

Specimen transported by a courier or mobile phlebotomist must be in a leakproof primary container. This container is put in a zip closure plastic bag containing absorbent materials such as paper towels and placed in a plastic or metal cooler or transport box which is the secondary container. Blood specimens can be placed upright in a rack that sits on top of absorbent material in the bottom of the transport box

The transport box must be closed to avoid spills and contamination in case it is jostled during transportation

The transport box should contain proper specimen data identification and the box should be placed on the floor of the vehicle behind the driver seat to avoid direct sun and ventilation

Specimens Transported locally are exempt from other US Department of transportation regulations, unless they are known or suspected to be infectious

out of area transportation

Diagnostic specimens that are transported out of the area by public transportation are covered by the US Department of transportation (DOT) and international air transportation Association (IATA) regulations for transportation of infectious substances.

DOT and IATA define two categories of infectious substances:

1. Biological substance category A: An infectious substance capable of causing permanent disability or life-threatening or fatal disease in normally healthy humans or animals

2. Biological substance category B: And infectious substance that does not need the criteria for category A. Includes laboratory specimens transported for diagnostic purposes. Requires all diagnostic specimens transported by public carriers to have triple packaging

Triple packaging requirements

-Specimens must be placed in a water tight container made of glass, metal, or plastic

-The individually wrapped containers must be placed in a leak proof secondary container, such as a sealed plastic biohazard bag strong enough to withstand leakage

-The primary and secondary containers must be placed in a sturdy third outer container made of wood, metal, or plastic

-Ice or dry ice must be placed outside the secondary container within the outer container or as in overpack

-Minimum required markings of the outer container include a UN3373 label with “Biological substance category B”, A Class 9 miscellaneous label of the package contains dry ice, the name, address, and phone number of the shipper and the receiver.

According to CLSI GP44-A4, serum or plasma should be physically separated from the cells-

As soon as possible unless evidence is conclusive that a longer contact time will not contribute to error in test results.

GP44-A4 also states that if an uncentriguged blood specimen Is sent to the laboratory from a blood drawing station, It must reach the laboratory in time for separation of cells and plasma or serum to occur with a time limit that protects the stability of the Analyte. If this cannot happen, the appropriate specimen processing must be done at the collection site and then delivered to the lab

CLSI guideline GP44-A4 recommends a time limit of less than two hours for separating serum and plasma from the cells for

catecholamines, homocysteine, lactic acid, and molecular tests targeting RNA such as HIV and HBV quantitative assays.

Studies cited by CLSI show that the two hour time limit also applies to

Glucose, Ionized calcium, Lactate dehydrogenase (LD or LDH), and potassium

Offsite locations from laboratory’s should have a small

Processing area where blood specimens for tests that are performed on serum or plasma can be centrifuged right away and the serum or plasma separated should be transferred to a leak proof secondary container for transport. These specimens should be stored properly until a courier service arrives.

Prompt delivery and separation minimize the effects of

Metabolic processes.

Examples include glycolysis. Unless chemically prevented by an additive such as sodium fluoride, Glycolysis continues in a blood specimen, lowering glucose levels until the serum or plasma is physically separated from the cells

Ammonia specimens

Levels of this increase rapidly at room temperature. Consequently, for accurate results, this specimen must be immediately placed on ice and transported STAT and separated from the cells within 15 min of collection

Coagulation specimens

The acceptable amount of time between coagulation specimen collection and testing depends on the test. However, it is best if all specimens for plasma based coagulation tests are processed as soon as possible after collection

Prothrombin time (PT) and partial Thromboplastin time (PTT or aPTT) tests should be performed within

4 hours of specimen collection

However, PT specimens can be held at room temperature, either centrifuged with the plasma in contact with the cells or uncentrifuged for up to 24 hours after collection, provided the tubes have not been open.

PTT SPECIMENS FROM PATIENTS WHO ARE NOT ON HEPARIN CAN BE HELD IN THE SAME MANNER, BUT ONLY FOR UP TO FOUR HOURS

PT or PTT Specimens that have been opened must be tested within four hours. If time limits for PT and PTT testing cannot be met-

The specimens platelet poor plasma can be frozen at -20c or below for up to 2 weeks or -70c for a longer storage.

PTT specimens for monitoring unfractionated heparin levels must be centrifuged within

1 hour of collection and tested within 4 hours of collection. If time constraints for samples from patients on unfractionated Harper cannot be met, The specimen should be collected in a CTAD tube

Glucose specimens in sodium fluoride tubes

Glucose specimens drawn in sodium fluoride tubes are stable for 24 hours at room temperature and for up to 48 hours when refrigerated at 4° to 8°C. However, inhibition of glycolysis may be adequate in specimens from patients with abnormally high platelet, RBC, or WBC counts. In addition, complete inhabitation of glycolysis by sodium fluoride can take as long as four hours, during which time glucose levels can fall as as much as as 10mg/dL, even in samples with normal blood cell counts.

Pediatric glucose specimens

Glucose specimens from newborn and pediatric patient should be tested as soon as possible because it is difficult to inhibit glycolysis in newborn and pediatric specimens. Micro collection devices with an appropriate anti-glycolic agent are available for collecting capillary glucose specimens from pediatric patients

Hematology specimens- blood smears

Blood smears made from EDTA specimens must be prepared within one hour of collection to preserve the integrity of the blood cells and prevent artifact formation due to prolonged contact with the anticoagulant

Hematology specimens- EDTA specimens for CBCs

Should be analyzed within six hours, but are generally stable for 24 hours at room temperature

Hematology specimens- EDTA specimens for erythrocyte sedimentation rate (ESR) determinations

Must be tested within four hours if left at room temperature or within 12 hours if refrigerated

HEMATOLOGY SPECIMENS- EDTA Specimens for reticulocyte counts

Are stable for up to six hours at room temperature and up to 72 hours if refrigerated

Molecular test specimens

Plasma preparation tubes for molecular testing such as hepatitis C or RNA Must be transported, processed, and tested as soon as possible because RNA substances are extremely unstable. If an RNA Test specimen must wait to be run in a batch, the plasma can be stored at 4°C, but only for 48 hours. If the plasma is not tested within that timeframe, it must be transferred from the PPT to an aliquot tube and frozen at -80°C. Collection tubes within inhibitors are available that prevent degradation of RNA and DNA. Use of these tubes instead of cold storage is recommended.

Microbiology specimens

Specimens collected for cultures should be transported to the laboratory as soon as possible. It is very important to properly transport and quickly process microbiology specimens to preserve any microorganisms in the specimens so they can be identified. After being accessioned, a sample of the specimen is transferred to culture media bottles or in the case of blood culture bottles, put immediately into an incubator

Urine specimens

These specimen should be transported to the lab promptly. The time between collection and transport is of great importance because the quality of the test results is dependent on the timelines of transport. If not, tested promptly, the components can change.

Ex-Cellular elements decompose, Bilirubin breaks down to biliverdin, and bacteria multiply, leading to erroneous test results.

Results that cannot be transported or analyzed promptly can be held at room temperature and protected from bright light up to two hours. Specimens help longer than two hours should be refrigerated.

UA and culture sensitivity testing should be refrigerated if immediate processing is not possible

URIN SPECIMENS FOR CYTOLOGY SHOULD BE EXAMINED IMMEDIATELY AFTER COLLECTION OR A PRESERVATIVE SUCH AS ETHANOL SHOULD BE ADDED TO AVOID DETERIORATION OF THE CELLS

Special handling

When blood leaves the body it is exposed to the effects of temperature and light that can negatively affect analytes. Specimens for analytes that are significantly affected require special handling to protect them.

body temperature specimens

Although most specimens can be transported at room temperature, Which for most labs is around 25°C, 77°F, Some specimens will precipitate or agglutinate if allowed to cool below body temp. These specimens need to be transported at or near the normal body temperature of 37°C. Most of these specimens require collection in a tube that has been pre-warmed to 37°C.

Small portable heat blocks that are kept in a 37°C incubator until needed are available for transporting body temperature specimens. The heat blocks hold this temperature for approximately 15 minutes after removal from the incubator. Temperature sensitive specimens that can withstand slightly higher than 37°C can be wrapped in an activated heel warmer.

Examples of specimens that need to be transported at body temperature

Cold agglutination, cryofibrinogen, cryoglobulin

Chilled specimens

Blood cell metabolism continues in a blood specimen after collection. Chilling the specimen slows down blood cell metabolism. It also protects thermolabile (altered or destroyed by heat) analytes. blood specimens that require chilling should be completely immersed in a slurry of crushed ice or water or put in a special cooling rack and either tested immediately or refrigerated on arrival in the laboratory.

Large cubes or chunks of ice without water added do not allow adequate cooling of the entire specimen. Contact with a solid piece of ice can freeze parts of the specimen, resulting in hemolysis and possible analyte breakdown

Examples of chilled specimens

ACTH, ammonia, catecholamines, gastrin, homocysteine, lactic acid, metanephrines, PTH, pyruvate

Specimens that must not be chilled

Some specimens are negatively effective by chilling during transportation. Most coagulation specimens must not be chilled because it can activate clotting factors and disrupt platelet function.

Potassium specimen should not be chilled because the cold inhibits glycolysis which provides the energy to pump potassium into the cells. With glycolysis inhibited, potassium leaks from the cells into the serum or plasma, artificially elevating levels in the specimen.

Arterial blood gas samples often also include testing for potassium. At one time ABG samples required chilling, but current recommendations state that ABG samples should not be placed on ice if analyzed within 30 minutes of collection.

Light sensitive specimens

Some analysts are photosensitive (Sensitive to light) and are broken down by light, resulting in falsely decreased values. The most common example is bilirubin, Which can decrease by up to 50% after one hour of light exposure.

Wrap specimens in aluminum foil. Amber tubes, biohazard bags, or light blocking transport containers can also be used.

Light blocking Amber colored micro collection containers are available for collecting infant capillary specimens.

Amber containers for urine specimen collection are also available

Examples of specimens that need to be protect protected from light

Beta-carotene, bilirubin, folate, vitamin A,B,C, urine porphobilinogen, urine prophyrins

Specimen processing

Most offsite drawing stations have processing areas where specimens are centrifuged and separated from the cells, this protects analyte Stability before the specimens are sent to the testing site.

Large laboratories typically have a specific areas which may be called the central specimen processing area where specimens are retrieved and prepared for testing. Here the specimens are identified, Logged, and Sorted by department, and type of process required

Special requirements must continue to be maintained throughout the processing and until the specimen is tested

OSHA required protective equipment

Processing specimens involves the possibility of exposure to blood-borne pathogens. Those who process specimens must wear personal protective equipment, which includes gloves and a fully closed fluid resistant lab coat. Wearing protective face gear such as masks and goggles with side shields or chin length face shields, or performing activities behind a bench top splash shield are required when manually opening or handling open specimen tubes or containers.

The most frequently cited reason for rejection of chemistry specimens is

Hemolysis, followed by insufficient amount of specimen, or QNS (quantity not sufficient).

The most frequent reason for rejection of hematology specimens is

Clotting

Clotting is also unacceptable for coagulation specimens.

Clotting in anticoagulant tubes, including plasma separator gel tubes is usually caused by inadequate mixing at the time of collection

Coagulation specimens have a critical blood to additive ratio and will also be rejected if the tube is overfilled or underfilled

Specimens that do not require further processing

If these types of specimens are sent to another type of facility, they are taken directly to a processing area where they are packaged and sent out. If they are tested on site they are sent to the specific area of testing in the lab

Specimens require no additional processing, such as centrifugation or separation: CBC, other whole blood specimens (cyclosporine, A1c, WB-lead analysis), cerebrospinal fluid, microbiology specimens, urinalysis specimens.

Specimens that require centrifugation

Tests that must be performed on serum or plasma.

The processing of these specimens have three phases: precentrifugation (after specimen collection and before centrifugation), centrifugation, and postcentrifugation (after centrifugation and before removal of serum or plasma).

Specimens that arrive from offsite locations may have already been centrifuged. If so-

They can proceed to the postcentrifugation stage after suitability requirements have been met.

Specimens for plasma tests are collected in anticoagulant tubes that prevent the blood from clotting, But they may still be centrifuged to separate the plasma from the cells. However, Because no waiting for the specimens to clot is involved, tubes for plasma tests, including those that contain gel can proceed to the centrifuge stage after meeting suitability requirements.

Precentrifugation

Non-additive, Clot activator, And gel-Containing tubes used for serum tests (ex-SSTs) Must clot for the serum to separate from the cells. They must not be centrifuged until clotting is complete. If clotting is not complete when they are centrifuged, latent fibrin formation may form a clot in the serum after centrifugation. Complete clotting takes 30 to 60 minutes at room temperature (22° to 25°C).

Specimens from patients on anticoagulant medication, such as heparin or warfarin, Specimens from patients with high WBC counts, And chilled specimens may take longer to do what?

Clot

Specimens from patients with coagulopathies (bleeding disorders) may take longer to

Clot or clot incompletely

Rotor

The part of the centrifuge that holds the tubes and spins. Carriers for holding the tubes are attached to the rotors.

Plasma tests that are collected in anticoagulant tubes may be centrifuged

Right away

most chemically tests have traditionally been tested on serum but STAT chem tests are sometimes collected in a green-top heparin tube to save time simply because plasma specimens

Can be centrifuged right away

Sedimentation

The ability of particles (cells in a tube of blood) in a suspension to settle to the bottom of a container overtime from the force of gravity

Spinning of a centrifuge creates force may times that of gravity causing-

Acceleration of sedimentation

RPM

refers to how fast the rotor is spinning

The force applied to the substance being centrifuged is called

Gravities or relative centrifugal force (RCF).

This force is a function of the rotation speed of the centrifuge and the rotation radius, and thus varies according to the size of the centrifuge

If the RCF required to spin the specimens is known, The radius of the centrifuge can be measured to determine the speed setting that should be used

Generally, centrifuging blood specimens at a setting that creates a force of 1000 G for 10 minutes will result in a good separation of serum or plasma

What tubes may require a force of 1000 to 1300 RCF

Gel tubes

It is crucial to balance specimen tubes when loading them into a centrifuge because

It can be counterbalanced with a tube of the same size filled with water to the same level. An unbalanced centrifuge may break tubes and caused tube contents to form aerosols.

If the centrifuge shakes, vibrates excessively, or is noisier than usual, it may Not be properly balanced

Before starting the centrifuge verify that the timer and speed are set

Correctly

A centrifuge time that is too long or a speed that is too high can hemolyze specimens.

A time that is too short or a speed that is too slow can result in incomplete separation of the specimen

You can easily check the speed of a centrifuge using a

Tachometer, An instrument designed for measuring RPM.

Serum or plasma removed from centrifuged specimens is placed-

in a plastic secondary tube for delivery to the testing area or transport to other testing facilities

The second tubes are often called transport tubes or aliquot tubes

Aliquot

A portion of a specimen used for testing. Aliquots Are typically created when multiple tests are ordered on a single specimen, and the tests are performed on different instruments or in different areas of the testing department.

To prepare an aliquot, transfer a portion of the serum or plasma from the centrifuged specimen into one or more tubes pre-labeled with the same identification information as the specimen tube.

Transfer pipettes should be used when transferring serum or plasma into aliquot tubes.

Platelet-poor plasma

Some coagulation tests require platelet poor plasma. To prepare platelet poor plasma, carefully transfer the top 3 quarters of plasma from the centrifuged specimen into an aliquot tube using a plastic transfer pipette. (never pour off plasma because it can introduce excess cells into the specimen). Cap the tube and spin it in the centrifuge a second time. Use a new pipette to transfer the plasma from the second spin into a new tube, being extremely careful not to disturb the platelets at the bottom of the tube.

In addition to patient ID label tubes with sample type and date and time of collection. Promptly freeze the specimen in an upright position after processing.

Storage

It is standard procedure to refrigerate most specimens after analysis

This chemistry test can be performed on separated serum or plasma for up to one week

Serum and plasma aliquots

Serum and plasma that has been separated from the cells should not remain at room temperature for longer than eight hours. If not tested before then they should be refrigerated at 2° to 8°C

Body fluids

Body fluids can be refrigerated after testing

Biobanking

Is a repository or storage facility where human biological samples can be stored, preserved, and cataloged for use in research for personalized medicine, for example

STAT and panic (critical) value test results are reported

Directly to the physician as soon as possible, typically by phone or paper. Panic values are significantly abnormal test results that could indicate a life-threatening situation that require immediate attention by the physician

Examples of panic values: sodium, potassium, magnesium, glucose, carbon dioxide, calcium, bilirubin, protime (PT), partial Thromboplastin time, hemoglobin, hematocrit, platelet count, WBC count

Reporting test results

the LIS generates an electronic report after the results are verified. The report is shared with medical records and the HIS, Where it can be accessed by the physician or other designated personnel