Rev 15 09 2017

THE IMPORTANCE OF IMPROVING THE TRANSPORT

OF BIOLOGICAL SAMPLES

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The pre-analytical variables, such as collection, han-dling, transport and storage of samples can alter the diagnostic results of the patients. The sample trans-portation is broadly acknowledged as one of the ma-jor factors that contribute to the attainment of reliable laboratory results in relation to both hospitalized and outside patients (1). International guidelines (2-3-4) and the UNI EN ISO 15189 certification specifically describe the recom-mendation for the pre-analytical procedures and un-derline the importance of the existence of strict crite-ria in order to evaluate the acceptability of the transport of primary samples. This short guide describes the recommendations of Italian and foreign laboratories for the correct

transport of samples, with reference to haematology, microbiology, mass spectrometry, etc., and highlights the crescent needs of a correct monitoring procedure of this pre-analytical phase (7). The main issues are illustrated in the table 1 and 2 (8). The correct management of the storage and transport activities of diagnostic samples makes the subject of recent regional resolutions (Dgr no. 3484

The importance of improving the transport of biological samples

The transport and the storage conditions of diagnostic samples are critical variables that increasingly impact on the quality and safety of the pre-analytical phase

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of 7 November 2006: laboratory medicine - that sets forth specific requirements for the institutional ac-creditation), regulations and reference standards (Circular Letter of the Italian Ministry of Health no. 3/2003, ISO 15189, ADR 2007, OMS, etc.) (12). Such indications highlight the need for intervention on the following critical issues:

Transport time (with particular reference to samples coming from local sites)

Transport temperature (with a specific focus on transports effected by means of cool boxes)

Transport conditions In case a sample is not transported at the adequate temperature, i.e. too hot or too cold and/or without respecting other specific conditions (i.e. keeping the primary container in a vertical position in order to fa-cilitate the blood clotting and to avoid the sample haemolysis), the results provided by the laboratory could be altered, causing an erroneous diagnosis and the wrong treatment of the patient (12).

Table 2

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The presence of haemolysis in a biological sample is mainly caused by haemolytic anaemia or in vitro haemolysis. The latter is the consequence of inappropriate activities of collection, processing and transport of the biological sample that can undermine the reliability of the results of many laboratory examinations and negatively affect the diagnosis and treatment of the patients.

As broadly described in literature, the in vitro haemolysis is the main cause of unfitness of the samples for in and out-patients, for routine and in urgency samples.

In general, the presence of haemolysis in the sample can affect the reliability of the results by:

1. the release of elements at a high intracellular concentration, that falsely increase the concentration of the same into the serum or plasma (i.e. potassium, transaminase, lactate dehydrogenase, LDL);

2. dilution and related spurious decrease into the serum or plasma of certain analytes that are little represented inside the red cells due to the exit of the intracellular water (i.e. sodium),

3. spectrophotometric interference in the determination due to the increase of the extinction (or “absorbance”), modification of the “blank value”, mainly for tests based on spectrophotometric reading at 415, 540 and 570 nm, that are wave lengths able to detect the free haemoglobin (i.e. gamma-glutamiltransferase, GGT);

4. a miscellaneous of other analytical interferences (i.e. spurious decrease of the bilirubin in the reaction of Jendrassik-Gróf due to the hemoglobin pseudoperoxidasic activity, increased creatine kinase activity further to the release of intra-erythrocyte adenylate kinase);

5. (v) release of substance at thromboplastin

activity (Table 3).

On the other hand, the magnesium behaviour is peculiar; indeed, besides the intra-erythrocyte concentration of this analyte is almost twice the plasmatic one, the increased release of magnesium by the red cells is over-balanced by the analytical interference in its determination caused by the presence of free haemoglobin, so that the global

BLOOD SAMPLES - HAEMOLYSIS

Dependent on the patient

1. fragile veins

2. difficultly traceable veins

3. Blood taking in a hematoma site

Dependent on the person in charge to take the blood sample

1. Experience

2. Failed attempts of blood taking

3. Trespassing of the vein

4. Vein lose during the blood sampling

Device used for taking the blood sample

1. Use of vascular catheters or butterfly needles

2. Use of reduced size needles (i.e. inferior to 23 Gauge)

3. Imperfect removal of the antiseptic solution from the

skin

4. Prolonged use of the tourniquet

5. Collection in inadequate containers

6. Inappropriate filling (insufficient or excessive) of the test

tube

7. Blood transfer from the syringe to the test tube

Treatment of the sample immediately after its taking

1. Excessive or inadequate agitation of the test tube

2. Inadequate mixing with the anticoagulant

3. Transport of the sample

4. Transport system (fitted with wheels, via couriers)

5. Transport conditions (mechanical trauma, duration, tem-

perature and humidity)

Treatment of the sample immediately before the analysis

1. Delay in the centrifugation

2. Centrifugation conditions of (speed, time, temperature)

3. Inefficient separation of plasma or serum from the

formed elements

4. Re-suspension of the sample after the centrifugation

Sample storage conditions

1. Re-centrifugation

2. Storage conditions (temperature and duration)

Table 3 - Main causes of in vitro haemolysis

The haemolysed sample is the most frequent cause of non conformity of the biological samples in the clinical laboratories representing the 40/70% of all non

conformity samples.

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Mid Modest Elevated

Spurious increase in serum or plasma due to the release

of analytes with an elevated intra-erythrocyte

concentration

Haemoglobin (Hb) - - ↑ or ↓

Potassium (K) ↑ ↑↑ ↑↑↑

Lactate Dehydrogenase (LDH) ↑ ↑↑ ↑↑↑

Aspartate Aminotransferase AST) ↑ ↑↑ ↑↑↑

Alanine Aminotransferase (ALT) -/↑ ↑ ↑↑

Creatinine (Crea) - - ↑

Dilution effect related to analytes with a low intraeritrocite

Albumin (Alb) - - ↓

Chloro (Cl) - - ↓

Glucose (Glu) - ↓ ↓↓

Sodium (Na) - - ↓

Chemical or Spectrophotometric interference

Alkaline Phosphatase (ALP) - - ↓

Bilirubin (Bil) - - ↑ or ↓

Creatin kinase (CK) - ↑ ↑↑

Iron - - ↑

ү-glutamyl-transferase (GGT) - - ↓

Lipase - - ↓

Magnesium - - ↓

Phosphore - - ↓

Urea - - ↓

Immunochemical determination (i.e. troponin) ↑or↓ ↑↑ or ↓↓

Release of substances with thromboplastin activity

Prothrombin time (PT) - ↑ ↑↑

D-dimer - ↑ ↑↑

Partially activated thromboplastin time - ↓ ↓↓

Antithrombin - - ↓

Table 4 - Grade of Hemolysis

Related to interference “>↑↑” / “>↓↓” or locally calculated and exceeding the limits of the desirable quality specifications defined on the basis of the biological variability intra and inter-individual or of the critical difference, the results shall be eliminated.

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effect is usually an under-estimation (6).

The causes of in vitro haemolysis start from the bed of the patient and extend through the whole process till the analysis. In general, such causes are due to: patient anatomic features (such as fragile or non-traceable veins), ability of the person in charge of blood sampling; device used for the blood sampling; treatment of the sample just after withdrawn; the delivery conditions to the laboratory; storage conditions, treatment of the sample before analysis (6).

As a precaution to avoid the in vitro hemolysis, it is important to transport the samples without mechanical agitations (for instance, avoiding the transport by pneumatic pipes) and to immediately separate plasma from cells (9).

There are several factors that are able to alter a blood sample, however transport system and storage are the most relevant. It is known that lengthy transport time, temperature variations and stretched out time gaps between the blood sampling and the analysis can alter the samples (12).

Temperature factor

Different temperatures are required for different parameter detections, however conservation temperature between 10°C and 22ºC is

recommended for most of the analytes.

Temperatures higher than 35 °C shall be avoided.

High temperature accelerates the deterioration of

the blood components.

Also the transport at temperatures lower than 0°C could cause hemolysis especially for those samples in direct contact with ice.

Room Temperature

Glucose decrease (for 2 hours at 23ºC – decrease of 10% of the concentration). The concentration decrease is higher in case of

pathologies such as the leukocytosis

Phosphate increase

Ammonium increase in 15min

Folate decrease in 30min

Vitamins B6 e B12 alteration

4ºC

Clotting factor VII becomes instable (the sample shall be kept at room temperature)

At temperatures lower than 4ºC or higher than 30 °C there is an increase in the release of potassium ions by the erythrocytes

The antibodies can affect the cell count performed in hematology as a consequence of their strong sensitivity to the storage temperature.

Literature studies highlight that storage duration at a certain temperature is a factor that influences the results i.e. if a blood sample is stored for more than 2 hours at 23°C the glucose concentration decreases of 10% (Fig.15-1).

Many analytes such as electrolytes and enzymes are not affected by alterations due to the transport up to 4 days. The major differences relate to hematocrit, MCV and bilirubin (13) (Fig. 15- 2).

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The appropriateness of the pre-analytical phase imply a convenient selection, an accurate collection and a prompt transport of all the samples. (11)

CEREBROSPINAL FLUID

The cerebrospinal fluid is a colorless and clear fluid that forms inside the nervous system’s cavities (ventricular cavities).

For chemical, bacteriological, immunological and cytological analysis of the cerebrospinal fluid, it is necessary to take a sample usually through the lumbar puncture, by inserting a needle in the space between the 4th and the 5th lumbar vertebra.(5)

Due to the delicacy and invasiveness of this fluid taking, it is important that the sample is immediately sent to the laboratory and that the sample temperature is monitored.

One of the microbiological tests performed on the cerebrospinal fluid samples is the Neisseria

meningìtidis, also known as meningococcus.

Meningococcus is a gram-negative bacterium known for causing the meningitis disease. It was discovered by Giovanni Battista Ughetti in 1880, then isolated and grown in vitro by Anton Weichselbaum. It only infects human beings; an animal carrier does not exist. It is the only strain of bacterial meningitis able to cause epidemics. Diplococcus is transmitted for inhaled. Meningococcus grows in rich culture media, such as blod agar and agar chocolate (these latter satisfy its nutritional requirements and neutralize the unsaturated fatty acids to which these bacteria are particularly sensitive).

At the first isolation, the growth is particularly lengthy and it is favored by the 5% of CO2. Outside the host, it is sensitive to UV radiations, drying and cold temperatures (lower than 24 °C: this shall be kept in mind in case of sampling for

diagnostic purposes).

This characteristics require that the samples to be analyzed are strictly monitored.

BLOOD CULTURE

Blood culture from peripheral vein

Transport conditions:

never cover the bar code of the test tubes with adhesive labels

insert the test in the computer system

send to the laboratory as soon as possible or, if this is not possible, store at 37° or at room

temperature for no more than 2 hours

transport the test tubes in the dedicated double compartment bags or in the air-tight stoppered rigid containers.

Blood culture from cvc

Transport conditions:

send to the laboratory as soon as possible or, if this is not possible, store at 37° or at room

temperature for no more than 2 hours

insert the test in the computer system

transport the test tubes in the dedicated double compartment bags or in the air-tight stoppered rigid containers

RESPIRATORY TRACT FLUIDS

Regarding the secretions of the lower respiratory tract, it is possible to proceed to the cultural test and to the isolation of fastidious/invasive and non-fastidious/non-invasive pathogenic micro-organisms. Specific tests, requiring the use of supplemental media and/or of particular techniques, can be performed based on clinic suspicion. For instance, such tests include: - Legionella spp, Nocardia spp, filamentous fungi, specific tests for cystic fibrosis (including B. cepacia).

The Good Laboratory Practices indicate the methods of sampling, storage and transport of this delicate samples (5):

SAMPLES FOR THE MICROBIOLOGY LABORATORY

The results of microbiology assays are highly affected

by the quality conditions of the sample

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SPUTUM

Transport conditions:

promptly send the material to the laboratory

insert the test in the computer system

not later than 1 hour from the collection, store the material at room temperature

it is possible to store the material in the container with transport medium in the fridge for 2-3 hours

delay in the delivery can determine “false negative” results (loss of fragile microorganisms) or “false positive” (overgrowth of contaminating flora)

in the laboratory, the sample will be always analyzed with a microscope in order to evaluate the grade of salivary contamination (not appropriate sample)

BRONCHIAL ASPIRATE

Transport conditions:

promptly send the material to the laboratory

insert the test in the computer system

should the immediate delivery be impossible, store at 4°C for not more than two hours

make sure that the sample is well closed

transport the sample in the dedicated double compartment bags or in the air-tight stoppered rigid containers

indicate that an antibiotic therapy is in progress

BRONCHOALVEOLAR LAVAGE

Transport conditions:

promptly send the material to the laboratory

insert the test in the computer system

should the immediate delivery be impossible, store at 4°C for not more than two hours

make sure that the sample is well closed

transport the sample in the dedicated double compartment bags or in the air-tight stoppered rigid containers

indicate that an antibiotic therapy is in progress

indicate that it is about bronchial cleaning and the quantity of physiological solution input

ENDOCERVICAL SWAB

The endocervical swab is one of the samples that can be analyzed in a microbiology laboratory. It consists of endocervix secretions sampling and allows the identification of some specific infections that affect the genital tract, such as the Neisseria

gonorrae, Mycoplasma and Chlamydia. Due to the delicacy of the samples, specific storage conditions have been outlined (17). The sample shall be rapidly transported to the laboratory. If this is not possible, the sample can be kept at room temperature for around 24 hours. Table 5

SURGICAL WOUNDS

Transport conditions:

send the material to the laboratory as soon as possible

insert the test in the computer system

should the immediate delivery be impossible, store the samples up to twelve hours at 4°C

CENTRAL VENOUS CATHETERS TIPS

Transport conditions:

it is possible to store the material in a test tube in the fridge for maximum of 48 hours, moistening the sample with some drops of physiological solution.

MIDSTREAM URINES

Transport conditions:

immediately close the container, carefully tightening the cap and avoiding to touch the inside

insert the test in the computer system

do not contaminate with the hands the inner edge of the container, open it only at the moment of the collection

deliver the material to the laboratory as soon as possible (it shall be treated within 2 hours from its emission), store it at room temperature; alternatively, store the urines in the fridge at + 4° C (not in the freezer) for maximum twelve hours.

Device storage temperature

Sample managment

4-8 °C Sample must be processed within 48 hours from the sampling (24 hours Neisseria gonorrae);

20-25 °C Sample must be processed within 24 hours from the sampling

TABLE 5 - endocervical samples

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The department of medicines evaluation and pharmacovigilance underlines the good practices for the transport of medicines (16):

All the players are required to strictly comply with the storage conditions outlined on the label.

The use of the terms “room temperature” is excluded, while specific indications are provided:

do not store over 30°C;

do not store over 25°C;

store between 2°C and 8°C;

do not freeze nor put it into the fridge

subzero – store in the freezer.

When the product is stable for six months at 40°C+ or -2°C, nothing shall be indicated on the label concerning the storage temperature. Therefore, occasional and temporary permanencies of products with no specific indications on the label, at a temperature of around 40°C are compatible with a storage regime that does not affect the safety or the efficacy of the medicines. In any case, this limit, corresponding to an extreme situation with respect to the storage temperatures, shall be avoided as a normal condition, preferring lower temperatures.

For the management optimization, it is considered that the storage and transport in unity, at a temperature lower than 25°C that can involve the medicines with a limit set at +25°C, +30°C and those without indications, shall be preferred.

In the distribution phase, it is appropriate the presence of a detection system, possibly external, that allows the verification of the temperature on the means of transport

DRUG TRANSPORT

Defined temperature thresholds must be strictly re-

spected.

If the parameters of storage and transport are not com-plied with there is high risk to irreversibly compromise

the active substances or drug composition.

A system that ensures the products quality is required

In the distribution phase, the presence of a detection system, possibly external, that monitors the transport

temperature is recomanded

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CYTOGENETIC SAMPLES

Cytogenetics is that branch of human genetics that studies the set of chromosomes of the individuals (karyotype) with the aim to identify any anomalies at the basis of specific pathologic conditions or of the reduced fertility. In order to perform this analysis, it is possible to use blood samples, chorionic villi or amni-otic fluid.

The container of the samples shall reach the cytoge-netics laboratory by means of a transport provided with a thermostatically controlled container able to grant that a range of temperature between 19 °C and 36 °C, which is required for the storage of the cytogenetic samples.

In case the shipment is performed through different means, it is necessary to grant the keeping of a range of temperature between 19 °C and 36 °C (11).

MOLECULAR BIOLOGY AND MASS SPECTROM-

ETRY

Methods of analysis such as genome sequencing, DNA microarray and mass spectrometry allow to ob-tain relevant quantities of data starting from a small volume of sample. For this reason, it is very im-portant to critically monitor the pre-analytical phases such as the collection, transport and storage of the sample as well as extraction and clean-up proce-dures (14), due to the fact that a single laboratory mistake can determine an incorrect diagnosis of a genetic disease.

Pre-analytics also strongly affects the serum samples used in the methods MALDI-TOF (15). It has been assessed that lengthy transport and room tempera-ture incubation generate low picks and subsequently alter the results.

OTHER SAMPLES

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[3] Clinical Laboratory Standard Institute (CLSI), Col-

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leading cause of unsuitable specimens in clinical la-

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[9] Abbas Ismail et. al, In vitro and in vivo haemolysis

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[11] MANUALE PRELIEVI E RACCOLTA CAMPIONI

Medicina di Laboratorio (AUSL Romagna)

[12] GUIDA AGLI ESAMI DI LABORATORIO ANALI-

SI (USLL 22 REGIONE VENETO)

[13] Samples: From the Patient to the Laboratory.

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Wisser · B. Zawta

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[15] Timmis JF et al. Preanalytic Influence of Sample

Handling on SELDI-TOF Serum Protein Profiles. Clin

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[16] SANITA’ TRASPORTO E DEPOSITO MEDICI-

NALI(Dipartimento di valutazione dei medicinali e

farmacovigilanza)

[17] PRESTAZIONI, MODALITÀ DI RACCOLTA,

CONSERVAZIONE ED INVIO DEI CAMPIONI(Unità

Operativa /Area U.O.C. MICROBIOLOGIA E VIRO-

LOGIA Azienda Ospedaliera S.Camillo-Forlanini Ro-

ma)

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