Routine histology samples

The lower lobes of both the left and right lung tissue were collected for the purposes of histological examination by forensic pathologists. Prior to sample collection, the forensic pathologist conducting the PM inspected the lungs for any macroscopic abnormalities, such as petechial haemorrhages.

Afterwards, sterile blades and scalpel handles were used to excise the entire lower lobes of both lungs.

The tissue samples were then placed in pre-labelled, 90 ml polypropylene tubes (CJ Labs, Cape Town) and transported within 10 minutes to the Forensic Pathology Division where 10% formalin

was added. The time interval between tissue sample collection and addition of formalin was not prolonged and as such prevented autolysis of tissue.

Histological preparation of tissue

To prepare slides for histological examination, 5 mm x 5 mm pieces of tissue from the deeper part of both lungs were cut separately using a sterile blade and placed into two different plastic cassettes labelled with the unique case number (WC/14/case_number/year). The tissue was secured in the cassette using a cassette cover and immediately placed into 10% formalin for 24 hours to facilitate tissue fixation. Afterwards, the cassette was placed in a Tissue-Tek VIP 5 Jr tissue processor where the tissue was processed as follows:

• Fixation – tissue was placed in 10% formalin (1 hour).

• Dehydration – after fixation, the tissue was immersed in increasing alcohol concentrations (70%, 85%, 96% and 100% alcohol) (3 hours).

• Clearing – the alcohol was then removed from tissue by placing tissue in pure xylene (3 hours).

• Impregnation – tissue was placed in paraffin wax to fill any gaps in the tissue (4 hours).

After completion of tissue processing, tissue blocks were embedded in paraffin wax; ensuring the correct orientation of the tissue to facilitate the cutting of tissue sections that include the histology being investigated. The tissue blocks were placed on an ice tray to rapidly cool down and solidify, thereby facilitating cutting of sections as soon as possible after processing. A MICROM microtome was used to cut sections of 3 µm thick. The sections were floated in a 60°C water bath to allow melting of the wax. Afterwards, the sections were picked up and placed onto an appropriately labelled glass slide. The slide was dried by placing it in a Scientific Series 9000 incubator at 77°C for 30 minutes, immediately placed into xylene to remove any remaining wax and the following steps were followed prior to staining:

• Hydration – to remove xylene from the sections by briefly placing the slides in decreasing concentrations of alcohol (100%, 96%, 85% and 70%).

• The sections were then rinsed in tap water and placed in haematoxylin for five minutes to stain the nucleus; followed by rinsing in tap water and placing in Scotts tap water to intensify the colour of the haematoxylin.

• Tap water was again used to rinse the sections and the sections immediately placed in eosin to stain the cytoplasm, after which they were rinsed in tap water.

• Dehydration – water was then removed by placing the slide in increasing concentrations of alcohol (70%, 85%, 96% and 100%).

• Clearing – alcohol was then removed by placing the slide in pure xylene and a cover slip placed on top of the section and DPX used as a mounting medium. The slide was air dried before being assessed under a light microscope by forensic pathologists.

3.4.3 Routine microbiology samples

Swabs from the left and right lungs, as well as heart, were collected for microbiological tests. For the lung swabs, a clean pair of forceps was used to lift the lower lobe of either lung, after which the lateral surface of the lung was sterilised by scorching. A sterile blade was used to make an incision on the scorched surface of the lung. A swab was inserted into the incision and rotated a few times to ensure maximum sample collection.

The lateral surface of the left ventricle of the heart was then sterilised by scorching, after which a sterile blade was used to make an incision into the left ventricle without disturbing the anatomy too much. A swab was inserted into the incision and rotated a few times. Soon after sample collection, the swabs were placed back into the original tubes and transported to the Microbiology Division for processing.

Swab processing

The swab samples were processed at the Microbiology Division of the NHLS laboratory at Tygerberg Hospital. After confirmation at the pre-analytical desk that the information on request forms and samples matched, the three swab samples were taken to the analytical desk for processing as follows:

• Three different agar plates (Chocolate, Blood and MacConkey) were appropriately labelled with the case number and sample type as it appeared on the request forms (Figure 3.1).

• Each swab sample was inoculated into each of the three different agar plates and a three-way streak conducted.

• The nine plates were incubated in a jacketed NUAIRE CO2 incubator at 36°C and 5.5% CO2 for 18-24 hours to allow for microbial growth.

If there was no growth evident after 18-24 hours, the plates were incubated for another 18-24 hours under the same conditions. However, if there was general growth of organisms, biochemical confirmatory tests like the Gram stain, BBL Sensi-Disc susceptibility test and Analytical Profile Index (API) were conducted. If biochemical tests could not identify the organism growing on a plate, a Biomerieux VITEK 2 XL machine was used to identify the organism.

Figure 3.1 The three agar plates, Chocolate (A), Blood (B) and MacConkey (C), used for the culture of microorganisms