Cytogenetics, i.e. the study of the structure and function of chromosomes, involves many analytical techniques. These include routine analysis of G-Banded chromosomes using cytogenetic banding techniques, as well as molecular techniques such as fluorescence in situ hybridisation (FISH) and comparative genomic hybridisation (CGH). In the UK, cytogenetic analysis is normally carried out by a National Health Service (NHS) Regional Genetics Service, where a fully comprehensive analysis of both constitutional and acquired abnormalities is carried out using a full range of standard and molecular cytogenetic techniques. The postnatal cytogenetic division at the NHS Greater Glasgow and Clyde region (NHSGGC) has implemented a banding and coverslipping method to improve automation and maximise laboratory efficiency. This article discusses the impact of the implementation of the new system.
by Louise Monkman, J. Colgan, L. Crawford, M. Campbell and G. Lowther
Postnatal cytogenetics
As part of postnatal cytogenetic diagnosis, blood chromosome analysis (or karyotyping) is undertaken to identify constitutional abnormalities (i.e. abnormalities which have been present since conception). Common reasons for referral for blood chromosome analysis are suspected Down syndrome or features indicative of another chromosome abnormality in a neonate; multiple congenital abnormalities; developmental delay in infants and short stature (especially in young girls). Individuals at risk of inheriting a chromosome disorder present in the family (i.e. an inherited structural rearrangement) are also referred, and such analyses may be carried out in adults experiencing infertility, or on the products of conception in cases of repeated miscarriage.
Abnormalities may be in the form of alterations in the structure of one or more chromosomes (chromosome rearrangements), or may consist of an entire extra or missing chromosome, i.e. aneuploidy. If the result of a rearrangement is the net gain or loss (duplication or deficiency) of chromosome material, i.e. an ‘unbalanced karyotype,) then phenotypic effects are likely. If the structural abnormality simply results in rearranged chromosome material (with no net duplication or deficiency) then the karyotype is termed ‘balanced’. In such an instance no adverse effects are likely although the individual carrying such a rearrangement may be predisposed to having children with a derivative ‘unbalanced’ chromosome constitution.
Blood chromosome analysis
Blood chromosome analysis involves slide preparation, banding techniques and subsequent analysis. Chromosome analysis can be performed on peripheral blood, cord blood, products of conception or skin biopsy specimens from individuals suspected of having a chromosome abnormality. Cells from bone marrow, blood, amniotic fluid, cord blood, tumors and other tissues can be cultured using standard cell culture techniques in order to increase the number of suitable cells. All specimens for chromosome preparation are grown and maintained in an aqueous growth medium. After cultures have been initiated, they are allowed to grow under specific conditions of temperature, humidity and pH until adequate numbers of dividing cells are present. A mitotic inhibitor, which arrests cell division, is then added to the culture [3].
After the cell cultures have grown the cells are harvested and the final step of the harvest procedure is slide preparation. Fixed cells from suspension cultures are dropped onto glass slides to allow for subsequent staining and analysis. Fixed cells from in situ cultures do not require this step because they are already attached to a coverslip. In the late 1960s banding techniques were developed which differentially stain chromosomes. These allow chromosomes of otherwise equal size to be differentiated, as well as the breakpoints and constituent chromosomes involved in chromosome translocations to be elucidated. Routine chromosome analysis refers to the analysis of metaphase chromosomes which have been banded using trypsin. This creates unique banding patterns on the chromosomes. After slides are prepared they are “aged” overnight at 60°C, or for one hour at 90°C, to enhance the chromosome banding. Slide making and banding can either be undertaken manually or automatically by means of an automated banding and coverslipping machine.
Optimising new instrumentation for improved efficiency
The postnatal cytogenetics division of the Greater Glasgow and Clyde (GGC) region of the UK NHS system had been experiencing difficulties with manual coverslipping in particular with the consistency and speed of the process. On average, the laboratory processes around 2800 samples a year, and it was recognised that it needed to implement automated procedures to improve reporting times, quality and success rates. The division needed a solution to improve the overall automation and throughput of blood samples to ensure efficient slide preparation, sample turnaround time and reporting of results.
To improve the efficiency of its banding and coverslipping procedures, the postnatal cytogenetics division chose to invest in new banding and coverslipping machines. Over the last year, several automated techniques have been introduced into the postnatal section, including a Thermo Scientific Varistain Gemini ES banding machine and a Thermo Scientific ClearVue automated coverslipping machine [Figure 1].
Banding procedure
The postnatal cytogenetics division initially bands a test slide to ascertain the optimal trypsin time. This is checked by a suitably qualified scientist and the time is adjusted as required. The batch of slides is then banded. For postnatal slides, it takes on average six minutes to band a batch of up to 20 slides. The banding machine is versatile and can run several programmes simultaneously, providing consistency and speed in the banding process. The machine is designed for high throughput, precision, safety, durability and flexibility in histology and cytology staining applications. For the postnatal division these features were key to improving the automation of the laboratory.
Coverslipping procedure
In an active laboratory, staining and coverslipping workflow patterns constantly fluctuate. The postnatal cytogenetics division uses the coverslipping machine to soak a rack of up to 20 slides in xylene and then remove each slide and apply mountant. Suction is used to pick up a coverslip and a pressure sensor is used to press it onto the slide. The mounted slide is then replaced in the rack where the slides remain until dry.
Results
The impact of these new technologies has been measured by comparing the quality, reporting times and failure rates over a six month period following the implementation of the new systems with the same data from a comparable six month period of the previous year.
The implementation of the new banding machine in the postnatal cytogenetics division has set new standards for the efficiency of slide banding. The overall workload of the technical laboratory staff involved in slide-making and banding has been reduced by around 50%, meaning that patients are being diagnosed and treated faster than previously. Prior to the implementation of the machine, four slides were prepared and manually banded from each blood culture. Automation of the process has reduced this to two slides per culture.
The implementation of the coverslipping machine has provided a solution to the workflow problems faced by the postnatal cytogenetics laboratory. The use of the coverslipping machine means that each slide is uniform in both the placement of the coverslip and the depth of the mountant, which has significantly improved procedures.
Banding quality enhancements
The banding quality has been compared for both urgent and routine samples before and after the implementation of the automated systems. Quality was evaluated using the G-banding Evaluation Score table from the UK Professional Guidelines [1]. A statistically significant increase was found in the quality of both urgent and routine samples before and after automation [Figure 2]. This may indeed be an underestimation of the improvement as samples exceeding a quality score of six may have been underscored.
Reporting time improvements
The UK Professional Guidelines [1] state that for postnatal samples, 95% of urgent samples should be reported within 10 days, and 95% of routine samples should be reported within 28 days. The decrease in reporting times and the increase in the percentage of cases reported within guideline times is evident. Statistical analysis shows that compared to pre-automation, the drop in reporting times post-automation is significant [Figures 3, 4].
Improvements in success rates
If a sample fails to meet the quality criteria as stated in the UK Professional Guidelines, it is required that the report states that the sample is ‘poor quality’ and a repeat sample should be requested. UK Professional Guidelines [2] state that the culture success rate should not be less than 97.5%.
The data [Figures 5, 6] illustrate that the success rate has risen and the poor quality rate has fallen for both urgent and routine samples.
Conclusions
Over the years increasing workloads, both in terms of volume testing and the numbers of analyses that a laboratory is expected to handle, have been a challenge to laboratories who are driven by financial pressures to make savings and provide a more cost-effective service. As a result, it is important that laboratories look for solutions to improve automation and throughput of samples. With today’s emphasis being placed on speed and reduced turnaround time, it is vital that laboratories have efficient laboratory processes in place to maximise efficiency and productivity. Today, many laboratories are required to supply a seamless 24 hour service, seven days a week. By using products that improve automation, the postnatal cytogenetics division of NHSGGC has been able to support and enhance its medical
laboratory workflows.
For the West of Scotland Regional Cytogenetics postnatal service the implementation of automated procedures has led to a significant improvement in reporting times, quality and success rates, and has also decreased the poor quality rate for both routine and urgent samples. Recent advances in laboratory automation systems have provided significant benefit for researchers who deal with large numbers of samples. By incorporating new automation solutions laboratory efficiency can be improved and results communicated more easily and effectively.
References
1. Professional Guidelines for Clinical Cytogenetics – General Best Practice Guidelines 2007; v1.02
2. Professional Guidelines for Clinical Cytogenetics – Postnatal Best Practice Guidelines 2007; v1.01
3. Gersen SL, Keagle MB (eds): The principles of clinical cytogenetics. 2nd edn. Humana Press, 2005
The authors
Louise Monkman, J. Colgan, L. Crawford,
M. Campbell, G. Lowther,
Cytogenetics Laboratory,
Duncan Guthrie Institute of Medical Genetics,
Yorkhill Hospital,
Glasgow, UK
