Quality Assurance in  Bacteriology and Immunology

Collection and Transportation of Clinical Specimens

THE LABORATORY diagnosis of an infectious disease begins with the collection of a clinical specimen for examination or processing in the laboratory ( the right one, collected at the right time, transported in the right way to the right laboratory). The laboratory with the help of well chosen techniques and methods for rapid isolation and identification confirms the diagnosis.

It has been observed that the most important and frequent factor affecting laboratory analysis, even in a well-functioning laboratory, is not the laboratory investigation itself but specimen preparation and errors in identification or labeling.

Proper collection of an appropriate clinical specimen is, hence, the first step in obtaining an accurate laboratory diagnosis of an infectious disease. Applying one’s knowledge of microbiology for the collection, transportation and storage of specimens is as important as it is in the laboratory. The interpretation of the observation may be misleading if the specimen is inadequate.

Guidelines for the collection and transportation of specimens should be made available to clinicians in a lucidly written format. These should be regularly revised depending upon the needs and the availability of new technology. There should be frequent (at least annual) consultations between physicians, nursing staff and the laboratory to update the guidelines. The guidelines must emphasize two important aspects :

1.      Collection of the specimen before the administration of antimicrobial agents

2.      Prevention of contamination of the specimen with externally present organisms or normal flora of the body.

General rules for collection and transportation of specimens are summarized in Table 7.1.

Table 7.1: Collection and transportation of specimens

7.1 Criteria for rejection of specimens

Several criteria can be considered by a laboratory on the basis of which the processing of a specimen may not be done by the laboratory. Such a decision must be made in light of the specific requested investigation. Laboratory investigations of a sample are a waste of time and resources if following criteria are not fulfilled :

     Missing or inadequate identification

     Insufficient quantity

     Specimen collected in an inappropriate container

     Contamination suspected

     Inappropriate transport or storage

     Unknown time delay

     Haemolysed blood sample.

7.2 Collection of specimens

Because of possible alterations in the specimen prior to measurement, the clinical state of the patient will not necessarily be reflected by the result of the laboratory investigation despite the correct laboratory performance unless the specimen is properly collected. Some of the important specimens and their proper collection and transportation methods are described here so as to ensure quality.

7.2.1 Blood

Whole blood is required for bacteriological examination. Serum separated from blood is used for serological techniques. Unless proper precautions are taken, venous blood gets contaminated with normal flora (both Gram positive and Gram negative) present on the surface of the skin at the site of venepuncture. Various studies have shown that 5-30% of positive blood cultures represent contamination with skin bacteria. To keep the chances of contamination low proper skin antisepsis is extremely important at the time of collection of the sample. Tincture of iodine (1-2%), povidone iodine (10%) and chlorhexidine (0.5% in 70% alcohol) are ideal agents. However, some individuals may be hypersensitive to iodine present in some of these. While collecting blood for culture following points must be remembered:

     Collect blood during the early stages of disease since the number of bacteria in blood is higher in the acute and early stages of disease

     Collect blood during paroxysm of fever since the number of bacteria is higher at high temperatures in patients with fever

     In the absence of antibiotic administration, 99% culture positivity can be seen with three blood cultures

     Small children usually have higher number of bacteria in their blood as compared to adults and hence less quantity of blood need to be collected from them (Table 7.2).

Table 7.2 : Volume of blood to be collected  at different ages

7.2.2 Pleural and peritoneal fluids

These need to be collected under similar aseptic conditions as are used for blood cultures. Their appearance should be indicated on the requisition form (see Table 7.3).

Table 7.3: Appearance and interpretations of fluids

7.2.3 Cerebrospinal fluid (CSF)

Examination of CSF is an essential step in the diagnosis of any patient with evidence of meningeal irritation or affected cerebrum. Almost 3-10 mL of CSF is collected and part of it is used for biochemical, immunological and microscopic examination and remaining for bacteriological or fungal examination. The following important precautions need to be taken for CSF collection and transportation:

     Collect CSF before antibiotic therapy is started

     Collect CSF in a screw - capped sterile container and not in an injection vial with cotton plug

     Do not delay transport and laboratory investigations

     Transport in a transport medium if delay in processing is unavoidable

     CSF is a precious specimen, handle it carefully and economically. It may not be possible to get a repeat specimen.

     Perform physical inspection immediately after collection and indicate findings on laboratory requisition form

The characteristics of the appearance of CSF are outlined in Table 7.4

Table 7.4: Appearance and interpretations of CSF

7.2.4 Sputum

Sputum is processed in the laboratory for aetiological investigation of bacterial and fungal infections of the lower respiratory tract. It is of utmost importance in the diagnosis of pulmonary tuberculosis. Sputum is frequently contaminated to some degree with commensals present in the oropharyngeal cavity. Some of these are potential pathogens of the lower respiratory tract ( eg pneumococci, Haemophilus influenzae).

7.2.5 Urine

Under normal circumstances urine is sterile. The lower part of the urethra and the genitalia are normally colonised by bacteria, many of which may also cause urinary tract infection. Since urine is a good growth medium for all sorts of bacteria, proper and aseptic collection assumes greater importance for this specimen.

For microbiological examination urine must be collected as a "clean catch-mid-stream" specimen.

Urine specimens should be transported to the laboratory within one hour for bacteriological examination, because of the continuous growth of bacteria in-vitro thus altering the actual concentration of organisms. A specimen containing 10³ bacteria/mL, two hours after collection may have 10⁵ bacteria/mL, when kept at ambient temperature. If transport cannot be immediately assured, the specimen should be refrigerated and processed within 24 hours.

One can monitor the overall acceptability of urine specimens being cultured within a laboratory by reviewing the quantitative results. There should be a clear biphasic distribution of colony counts, with the majority of specimens containing either <10³ or > 10⁵ viable cells per mL. Relatively few urine specimens should contain micro-organisms in the range of 10⁴ to 10⁵ bacteria per mL, and the number with colony counts between 10³ and 10⁴ bacteria per mL should be held to a minimum. One can routinely monitor the results of urine colony counts on specimens collected from different clinics or wards within an institution. The results can be plotted periodically in order to detect sudden or gradual changes in the number of equivocal or positive specimens. Such an endeavor would indicate the time and place for another educational drive concerning the methods for collecting urine specimens.

7.2.6 Stool

Faecal specimens for the aetiological diagnosis of acute infectious diarrhoeas should be collected in the early stage of illness and prior to treatment with antimicrobials. A stool specimen rather than a rectal swab is preferred.

Specimens for bacteriological examination should be transported to the laboratory and processed within a few hours. In case of delay the specimen should be refrigerated. If longer delays cannot be avoided, a special transport medium should be used. In such media, pathogens survive for up to one week even at room temperature, although refrigeration is preferable. Various media that are in use are shown in Table 7.5. In case a stool specimen is intended only for parasitological examination (ova and cysts), it can be stored at room temperature.

Table 7.5: Transport media for stool specimens

7.3 Transportation of specimens

Specimens to be sent to other laboratoriesrequire special attention for safe packing of the material. Guidelines are usually issued by the national authorities and the same should be strictly followed. For hand carried transportation over a short distance, the specimen should be placed upright in appropriate racks. For long distance transportation, it should be placed in three containers, i.e:

     A primary container which has the specimen and is leakproof with a screwcap.

     A secondary container which is durable, waterproof and made of metal or plastic with a screwcap. It should have enough absorptive material to absorb the contents of the primary container should the latter break or leak. On its outside the details of the specimen should be pasted.

     A tertiary container is usually made of wood or cardbox. It should be capable of withstanding the shocks and trauma of transportation. Dry ice can be kept between this and the secondary container along with sufficient absorbents and provision for the escape of carbon dioxide to prevent a pressure buildup inside (Fig 7.1 ).

Fig 7.1: Transportation container

In general, most specimens should be processed in the laboratory within 1 to 2 hours after collection. In practice, a 2-to 4-hour time limit is probably more practical during a normal working day. The laboratory must be organized to permit processing of the specimens as soon as they arrive, and the collection of most specimens should be limited to the working hours of the laboratory. However, some arrangements must be made to allow for the initial handling of the few specimens that have to be collected outside of the laboratory’s working hours.

A continuous effort must be made in order to ensure proper collection and transportation of clinical specimens. Full cooperation of nursing staff and others concerned with specimen collection is required and can be achieved once they are made aware of the principles involved and the significance of what they are being asked to do.

The initiation of a programme to detect misuse of the laboratory’s facilities might also be considered. One would carry out such a programme by monitoring the number of tests performed on each patient. When the same type of procedure is requested repeatedly within a short period of time, some gentle inquiries might be made in order to determine whether the procedures are truly relevant to the care of the patient. Although it is impossible to establish any firm guidelines for monitoring the clinical relevance of the repetitive requests for investigations, frequent consultations with physicians will facilitate better coordination and reduce work-load of the laboratory.