Fundamentals of Histocompatibility & Immunogenetics —
Training activity: 3

Details

Isolate, quantify and store lymphocytes according to all local protocols

Type

Entrustable training activity (ETA)

Evidence requirements

Evidence the activity has been undertaken by the trainee repeatedly, consistently, and effectively over time, in a range of situations. This may include occasions where the trainee has not successfully achieved the outcome of the activity themselves. For example, because it was not appropriate to undertake the task in the circumstances or the trainees recognised their own limitations and sought help or advice to ensure the activity reached an appropriate conclusion. ​

Reflection at multiple timepoints on the trainee learning journey for this activity.

Considerations

• Principles of lymphocyte isolation methods
• Procedures available for T and B cell isolation
• Lymphocyte identification by microscopy
• Principles involved in lymphocyte cryopreservation
• Infection control

Reflective practice guidance

The guidance below is provided to support reflection at different time points, providing you with questions to aid you to reflect for this training activity. They are provided for guidance and should not be considered as a mandatory checklist. Trainees should not be expected to provide answers to each of the guidance questions listed.

Before action

What does success look like?

• Identify what is expected of you in relation to isolating, quantifying, and storing viable lymphocytes from blood samples.
• Consider how the learning outcomes apply, specifically in relation to performing the relevant laboratory investigations and employing safe working practices to prepare and handle samples.
• Discuss with your training officer to gain clarity of what is expected of you in relation to minimum acceptable cell yield/viability thresholds, use of specialized separation media, and long-term storage requirements.

What is your prior experience of this activity?

• Think about what you already know about cell isolation techniques, working aseptically, and using hemocytometers or automated cell counters for quantification.
• Consider possible challenges you might face during the activity, such as poor cell yield, low cell viability, or unexpected contamination during the isolation process.
• Recognise the scope of your own practice for this activity i.e. know when you will need to seek advice or help, and from whom. You will need to seek advice from your Training Officer when required, for example if the cell viability is below the minimum required standard after isolation, necessitating a discussion on whether the sample is suitable for storage and subsequent use.
• Acknowledge how you feel about performing a delicate procedure requiring high technical precision and aseptic technique.

What do you anticipate you will learn from the experience?

• Consider the specific skills you want to develop, such as refining density gradient centrifugation techniques or performing accurate manual cell counts.
• Identify the specific insights you hope to gain into the factors that influence cell viability and recovery during lymphocyte preparation.

What additional considerations do you need to make?

• Consult actions identified following previous experiences of cell culture or isolation procedures.
• Identify important information you need to consider before embarking on the activity, such as reviewing the specific protocols for different separation media, calculating reagent concentrations, and confirming the status of liquid nitrogen storage.

In action

Is anything unexpected occurring?

• Are you noticing anything surprising or different from what you anticipate whilst isolating, quantifying, and storing lymphocytes?
• Are you encountering situations such as:
  • The lymphocyte separation process resulting in significantly fewer cells or lower viability than anticipated?
  • You face unexpected difficulties during cell quantification e.g., clumping, excessive debris, or instrument errors?
  • Issues arising with essential reagents e.g., separation media, viability stain or cryogenic storage conditions?

How are you reacting to the unexpected development?

• How is this impacting your actions? For example, are you responding to the situation appropriately? Are you adapting or changing your approach to isolation or quantification technique?
• Consider the steps you are taking in the moment, such as:
  • Immediately documenting the low yield/viability and consulting the Training Officer regarding the viability threshold for downstream use
  • Implementing a troubleshooting step, such as recounting the cells or performing viability checks to handle quantification difficulties
• How are you feeling in that moment? For instance, are you finding it difficult to handle the delicate quantification steps under time pressure? Is it affecting your confidence in performing the task independently?

What is the conclusion or outcome?

• Identify how you are working within your scope of practice. For example, are you successfully verifying the integrity of the storage environment after performing routine checks? Or are you needing support because the unexpectedly low cell viability requires senior guidance on whether the sample should be accepted for storage?
• What are you learning as a result of the unexpected development? For example, are you mastering a more reliable technique for density gradient separation? Or gaining insight into the causes of low cell viability during processing?

On action

What happened?

• Begin by summarising the key steps you took when isolating, quantifying, and storing lymphocytes.
• Consider specific events, actions, or interactions which felt important, such as how you performed density gradient separation or quantified cells using an automated counter or haemocytometer.
• Include any ‘reflect-in-action’ moments where you had to adapt to the situation as it unfolded, for instance, immediately adjusting the dilution factor for quantification when initial counting suggested poor yield or seeking immediate advice on unexpected contamination.
• How did you feel during this experience, e.g., did you feel meticulous about aseptic technique or concerned about cell viability?

How has this experience contributed to your developing practice?

• Identify what learning you can take from this experience regarding lymphocyte processing and handling according to protocols. What strengths did you demonstrate, e.g., adherence to aseptic technique?
• What skills and/or knowledge gaps were evident, e.g., troubleshooting poor cell yield causes or managing cryogenic storage documentation?
• Compare this experience against previous engagement with similar activities – were any previously identified actions for development achieved? Has your practice improved in following local protocols accurately?
• Identify any challenges you experienced, such as low cell viability, quantification errors, or needing advice on scope of practice regarding minimum acceptable viability thresholds, and how you reacted to this.

What will you take from the experience moving forward?

• Identify the actions or ‘next steps’ you will now take to support the assimilation of what you have learnt, including from any feedback you have received, with regards to refining your cell isolation technique and improving adherence to protocols.
• What will you do differently next time you approach lymphocyte processing, for instance, by proactively verifying reagent expiry dates before starting isolation?
• Do you need to practise any aspect of the activity further, such as complex separation techniques or key learning outcomes related to employing safe working practices?

Beyond action

Have you revisited the experiences?

• How have your subsequent experiences of isolating, quantifying, and storing lymphocytes since completing this specific training activity led you to revisit your initial approach or decisions during that activity? For example, how a subsequent cell-based assay result that failed due to poor cell viability forced you to re-evaluate the diligence of your initial isolation and quantification steps during your first attempt at this training activity.
• Considering what you understand about cell recovery, viability thresholds, and cryopreservation techniques now, were the actions or considerations you identified after your initial reflection on this training activity sufficient? How have you since implemented or adapted improvements in your cell counting accuracy or isolation steps based on further learning and experiences? For example, how you proactively implemented a double-check of cell counting accuracy with a peer based on further learning.
• Has discussing best practices for lymphocyte isolation or troubleshooting common issues like poor cell yield with colleagues, peers, or supervisors changed how you now view your initial experience in this training activity? For example, how professional storytelling with a senior colleague about a time when a batch of cells was lost due to incorrect cryopreservation media refined your understanding of the critical nature of adhering to reagent preparation protocols during this complex process.

How have these experiences impacted upon current practice?

• How has the learning from this initial training activity, in combination with subsequent experiences of lymphocyte isolation, contributed to your overall confidence and competence in preparing viable and quantified lymphocyte samples, particularly in preparing for general observed assessments (DOPS or OCEs) involving cell handling? For example, how your accumulated ability in sterile technique and cell counting now enables you to manage complex cell preparations efficiently during an assessment.
• How has reflecting back on this specific training activity, combined with everything you’ve learned since, shaped your current approach to lymphocyte isolation and storage? How does this evolved understanding help you identify when lymphocyte quality is suboptimal, or the isolation process requires expert intervention and when this is beyond your scope of practice? For example, how your evolved approach means you now routinely seek expert intervention immediately when the required cell viability is below the minimum threshold, recognising this requires senior guidance on sample usability.
• Looking holistically at your training journey, how has this initial lymphocyte preparation experience, revisited with your current perspective, contributed to your development in meeting the learning outcomes related to performing relevant investigations and employing safe working practices? For example, how this foundational experience has supported your development in transferable skills such as sterile technique and cryopreservation that will be valuable in future roles or responsibilities.

Relevant learning outcomes

#	Outcome
# 3	Outcome Perform the relevant laboratory investigations for a range of patients referred to an H&I laboratory.
# 6	Outcome Practice in accordance with quality and accreditation standards.
# 7	Outcome Employ safe working practices to maintain laboratory equipment, prepare, handle and store laboratory reagents and patient samples.