Cellular Pathology - University Hospitals Birmingham

Due to the wide range of cellular pathology specimens several key container types exist as below.  The size of the specimen container must be appropriate to the size of the specimen.

To ensure that cases are not delayed within the laboratories please make sure that the request form is correctly completed and all sections are filled in properly. Once received urgent cases will be highlighted by the specimen reception and prioritised appropriately in the departmental workload. 

Please note many of the technical preparation aspects of cytology may take place at the Heartlands Hospital Cell Path Laboratory.

Intra-operative Frozen Sections

The department provides a rapid diagnostic service for intra-operative tissue specimens. Requests for frozen section should be telephoned to the histopathology laboratory (at QE site 0121 371 13314 and at BHH site 0121 424 3188 ) in advance of the procedure being undertaken, preferably on the day prior to surgery. Where this is not possible, e.g. an incidental finding during surgery, the request should still be telephoned to the laboratory prior to the sample leaving theatre. When calling the laboratory please ensure that you have the following information available as it will be asked for by the person receiving the call: 

Tissue taken for frozen section should be placed into a suitably sized container without any form of fixative (i.e. dry) and sent in a secondary bag/container to Cellular Pathology without delay.  The transport of the fresh material is arranged by the requesting clinical team, typically using a UHB Hospital porter.  Theatre books should be used to record the audit trail from theatre and safe receipt in the laboratory.  The process from specimen receipt in the laboratory to verbal report issued takes 15 to 30  minutes however this time may be shortened or lengthened slightly based on the nature of the sample received.  High risk biohazardous tissues are not suitable for frozen sectioning unless absolutely clinically required. Examples of these include tuberculous lesions, specimens from patients with viral hepatitis and specimens from patients who are HIV positive. If there is any doubt as to whether the specimen is suitable or not please contact the laboratory and ask for the advice of a Consultant Pathologist.

MOHS

Mohs, is microscopically controlled surgery used to treat common types of skin cancer. During the surgery, after each removal of tissue and while the patient waits, the fresh tissue is snap frozen and sectioned for microscopic examination. The examination informs the decision for additional tissue removal.

The process from specimen receipt in the laboratory to verbal report issued takes no longer than 15 minutes per sample block however complex specimens with multiple samples will take additional time.

Mohs clinics are booked with the Department to ensure that there are clinical and technical staff are available to complete the test.  Mohs surgery is carried out at Solihull and QE Hospital sites.

Neuropathological Smears

The Department provides a rapid diagnostic service for intra-operative brain tissue specimens. Requests for neuro smears should be telephoned to the laboratory in advance as per intra-operative frozen sections.

Tissue taken for neuropathological smears should be placed into a sterile universal container (UC) without any form of fixative (i.e. dry) and sent to Cellular Pathology without delay. These specimens are often tiny and so any delay in receipt may lead to artefactual damage which can be detrimental to any further laboratory processing.

The process from specimen receipt in the laboratory to verbal report issued takes  15 - 30  minutes however this time may be shortened or lengthened slightly based on the nature of the sample received.

Small Biopsy Specimens

The Department provides both a routine and urgent diagnostic service for small biopsy specimens.

Specimens should be placed into 60 mL pre-filled containers of neutral buffered formalin.

• Wherever possible small or endoscopic biopsies should be placed into mini biopsy or microcassettes cassettes (small yellow cassettes) before being placed into the neutral buffered formalin pots. Supplies of cellsafe cassettes can be sourced from the Cellular Pathology Specimen Reception.

• Breast or other fine cores may be placed onto white cards before being placed into neutral buffered formalin pots. Please ensure the cores are stretched out linearly prior to fixation as knotted cores are not easy to separate out in the laboratory.

Please note that biopsies are delicate and should be handled with care, avoid the use of forceps wherever possible to prevent trauma artefact to the tissue.

Urgent Biopsy Specimens (Rapid Paraffin Service)

The Department provides a rapid paraffin service for clinically urgent biopsy specimens; a report can be issued on the same day that the specimen arrives in the department.  It should be noted however that not all specimens are suitable for rapid processing. In general terms only liver, renal and cardiac biopsies are processed in this way. Other specimens may be suitable however advice should be sought from the Histopathology Manager (0121 371 3352) or a trimming room senior Biomedical Scientist (0121 371 3314) prior to sending other tissue types.

Tissues for the rapid paraffin service must be received by the department no later than 13:30. Please ensure that the name and bleep number of the requesting clinician is clearly indicated on the request form to allow for the verbal report to be issued.

Specimens received after 13:30 will not be processed urgently and will be placed onto the “normal” biopsy run later in the day thus same day reporting will not be possible.

Resection Specimens

The department provides a routine and urgent diagnostic histopathology service for larger surgical resection specimens. Tissue should be placed into appropriately sized containers which will allow for the specimen and at least ten times its volume in fixative (i.e. neutral buffered formalin) to be contained.

DO NOT use containers which are too small to allow for this volume of fixative to be used or squash specimens into small pots. Specimens should be free-floating in fixative not pressed up against the sides of the container as this leads to distortion of the tissue and may make further processing unnecessarily difficult.

Cytopathology

The Department provides the diagnostic cytopathology services for the Trust including fine needle aspirations, endoscopic brushings and washings, EUS-FNAs , serous fluids urines and CSF.  Routine specimens should reach the laboratory before 16:30 so that they can be processed within normal working hours. Specimens collected out of laboratory hours should be stored refrigerated until the next morning on which the laboratory is open.  Cytology sample that do not meet the minimum data requirements on request form or sample, are processed, to ensure that sample integrate is maintained. Details of missing data are added to the report.  Request for Consumables and Request Forms

Sample containers for use in cytology and request forms can be order by contacting the Cytology Department; they can be collected from the Cellular Pathology reception at Heatlands or QEHB sites.  Cytology samples which do not meet the MDS will not be returned.  They will be processed in order to preserve the diagnostic material while attempts are made to contact the sender.  These samples will not be reported until adequate labelling of specimen and request form has been undertaken.Minimal delay between collection and receipt by the laboratory is necessary in order to prevent degeneration of cellular components and consequent loss of diagnostic value. All cytological specimens are potentially biohazardous and must be transported in leak proof containers enclosed in sealed bags. Specimens sent to the laboratory via the SDS should be placed in specimen bags and lids on the all containers should be securely closed. 

Specimens from patients with known or suspected tuberculosis, HIV, viral hepatitis or other transmissible disease should be labelled clearly.  Cytology DO NOT except or handle any specimens that are ? prion disease or CJD.

Specimens should be received as either pre-prepared slides, fresh unfixed samples in white- topped universal containers, or suspended in CRR.  Equipment used to collect the specimen such as brushes or drains are inappropriate.

Sputum - A series of three early morning ‘deep cough’ specimens should be collected on three consecutive days for maximum sensitivity. Post physiotherapy and post bronchoscopy specimens are suitable but should be clearly identified as such.

Serous Fluids - 50mls or the whole volume if less is aspirated should be sent in sterile universal containers. Include any tissue fragments or clots. Do not add fixative of anticoagulant. Drain bags are not suitable for transporting specimens and should NOT be sent.

Cerebrospinal Fluid (CSF) - Rapid processing is essential to preserve cells in CSF. These specimens should be sent to the laboratory within one hour.  ALL CSF SPECIMENS SHOULD REACH THE LABORATORY BY 16:30 AT THE LATEST TO ALLOW PREPARATION.

Urine - A representative aliquot of a maximum of 25ml of urine is sufficient for cytology processing, Ensure that this is not the first sample of the day.  Catheter urine and bladder washings are also acceptable but please mention this on the request form. Mid-stream urine samples are not suitable for cytology because they contain few cells.

Endoscopic Brushing - Immediate fixation is important and all slides must be labelled with PENCIL with the patient’s name and hospital number before the smears are made.

Fine needle aspiration - Unless the clinician is experienced in this procedure, including making good quality smears, it is recommended that they contact the laboratory for advice before beginning the procedure. Smears should be made and the needle rinsed out in cytorich red fluid. For ENT specimens in particular it is important that smears are sent as well as needle washings. Only prepared smears and needle washings are accepted. The sending of needles sheathed or otherwise is strictly forbidden.

EUS-FNAs - Please send all the specimen in cytorich red fluid

Cervical Smears - Cervical smears are NOT accepted in nor prepared by cytopathology at QEHB. Please send any cervical smear specimens to the Cytopathology Laboratory at Wolverhampton's Black Country Partnership Laboratories.

Reporting

Once specimens are reported and authorised by the laboratory they can be accessed on the intranet by appropriate staff. In addition a typed copy will be sent to the destination specified on the request form (FOR EXTERNAL USERS ONLY). If the specimen is needed for a particular MDT or clinic, then please state this on the request form. Urgent reports can be issued verbally if necessary to a suitable member of medical staff. Please put a mobile telephone number or a bleep number (that will be answered immediately) on the request form. Telephone reports will only be issued on request via telephone.

Muscle Biopsy Service - specific requirements

Muscle biopsies are performed as part of the investigation of a clinically suspected neuromuscular disorder when other less invasive tests have not provided a firm diagnosis.  The Muscle Biopsy Service receives muscle and nerve samples from the whole of the West Midlands region and occasionally from elsewhere in the UK.

This Department offers the following investigative techniques:

• Muscle Histochemistry methods on frozen sections
• Muscle immunocytochemistry on frozen sections.
• Electron Microscopy.
• Histology and Muscle immunocytochemistry on paraffin wax sections.
• Arrangements can be made to send tissue away for DNA analysis.

 If required samples can be referred to specialist centres for further investigation

• Metabolic tests.
• Mitochondrial Assays and MtDNA.
• Protein analysis for adult Limb Girdle Muscular Dystrophies.
• Rare paediatric dystrophies

If a member of the muscle biopsy team is required to collect a nerve or muscle biopsy from QEHB theatres there should be a delay of no longer than 30 minutes between the biopsy being taken and the sample are being placed into fixative for electron microscopy.

For QEHB service users - Please call the lab to request collection as soon as possible once the specimen is available.  Prior to collection muscle should be kept in a dry universal container on water ice (ice can be supplied by the Muscle Lab) and the nerve should be kept in a dry universal container at room temperature.  A copy of the Muscle Biopsy request form noting the time the biopsy was taken should be completed by the requesting clinician. 

For external service users - Medical or secretarial staff from the requesting centre liaises directly with the Muscle Lab to book a biopsy.   A Senior Biomedical Scientist will travel to the hospital to collect the biopsy which is received fresh.  Clinical details are preferably received in advance of the biopsy from the requesting medical staff but relevant information may be transcribed from the patient’s notes onto the Muscle Biopsy Request Form by the Scientist collecting the biopsy.  The Scientist decides which tests will be required on the basis of this information.  The biopsy is cut up and fixed for certain tests on site, the rest of the specimen is brought back to the lab on ice and some is frozen in liquid nitrogen and the remainder allocated for further tests. 

Muscle Biopsy Service :

Cases can be requested as urgent at the time of the biopsy or by email/telephone; the pathologist will relay results back to the clinician via email/telephone.  

• Muscle report  – 4 weeks from receipt to report authorisation.
• Nerve reports – 4 weeks from receipt to report authorisation.
• The current turnaround time for cases referred by the Department to specialist centres is 2-3 months depending on tests requested.

Electron Microscopy Service - specific requirements

The Electron Microscope Unit is used routinely at magnifications of between 1500 and 70,000 times to examine the ultrastructure of cells and their surroundings.  The EM unit receives approx. 800 samples a year, the majority of which are renal, but about 100 muscle and 30 nerve biopsies are also collected.  Other specimens include cardiac biopsies, occasional skin biopsies and tumour samples.

Specimens from outside the department requiring EM should be sent in a small sample container (e.g. bijou or Eppendorf tube) containing at least 2 mL of an EM fixative.  A glutaraldehyde based fixative such as 2.5% glutaraldehyde in phosphate buffer is ideal but 10% buffered formalin is also acceptable.  In larger tissue samples individual pieces should be no larger than 3 mm cubed and should be completely submersed in fixative.  The optimal size of diagnostic tissue requiring processing for electron microscopy is as follows: the largest dimension should be no greater than 5mm and the smallest dimension no less than 1mm. Any specimen with the smallest dimension less than 1mm may not survive tissue processing.

For QEHB service users - Renal biopsies are delivered to the Cellular Pathology Specimen Reception and EM samples are taken in the EM Lab. 

For External service users - package specimens for EM in firmly closed leak proof containers, together with sufficient absorbent material to contain any liquid, in sealed bags. The sealed bags are placed securely in a small padded envelope with the address label as given above under key information.  The EM unit makes use of the Histology request form for both internal and external users.   A copy of the light microscopy report is also required.  

Please note that we support the service by outsourcing when required to the specialist EM facility at Leicester Royal Infirmary.  It is fully accredited by United Kingdom Accreditation Services (UKAS) ISO 15189, reference number 8608.

Advice on the service or requirements can be obtained from the EM lead biomedical scientist This email address is being protected from spambots. You need JavaScript enabled to view it. or from the electron microscopy laboratory team on 0121 371 5720.

Laboratory Turnaround Times

The Department monitors turnaround times on a regular basis and reviews significant delays or service issues within the Trust's Divisional Diagnostic Performance monthly meetings.  7, 10 and 14 day Cellular Pathology turnaround target reporting times are reported to Trust service users, broken down by clinical specialty for biopsies and for cancer tracking cases for each month.  Specific turnaround data is available to service users upon request in addition to UHB internally available histopathology intranet dashboards.  The agreed routine TATs for each specialty is 14 days to first reported for greater than or equal to 90% of cases, within tolerance = 75% - 90% reported in 14 days.  Targets for reporting are not consistantly met, but of note is some more complex haematopathology and urology cases  may require additional work e.g.  immunohistochemistry or molecular pathology and this does add to reporting times for complex cases.  

In addition the Department actively monitors outstanding cases, notifying reporting pathologists that cases are still unreported.  This and actions to increase lab and consultant capacity continue to support the delivery of improved TATs for service users.

Muscle Biopsy Service :

• Histochemistry results is 7 days
• The current turnaround time for cases referred to specialist centres is 2-3 months depending on tests requested.

Electron Microscopy :

• Renal and cardiac turnaround times are approx. 10 to 14 days, TATs may be longer with a small specialist EM team
• Muscle and nerve biopsies approx. 10 to 14 weeks.

Referral Laboratories and second opionons

For some complex cases outside, second opinions may be requested  internally within UHB but also externally to support the diagnostic report and this may add to the number of days before a final diagnostic report is issued.   List of key contacts we currently use for specialist second opinions and referral labs used for testing. 

Application of the CV to the clinical relevance of Diagnostic Semen Analysis (DSA)

The Coefficient of Variation (CV) was calculated for each parameter for DSA and used as the measurement of uncertainty associated with each parameter. Each parameter’s CV is as follows:

 The uncertainty is calculated by dividing the analysis result by 100 and multiplying by the associated CV. The range is calculated by +/- the uncertainty from the result.

 The uncertainty could influence the fertility pathway for patients who have received results close to the clinical decision values for each specific parameter. Examples include:

Motility – Uncertainty = 2.2%

This informs us that if the laboratory reports a motility result of 30% then it will have a range of 30% ± 2.2 (27.8 – 32.2%).

Concentration – Uncertainty = 1.4 x 10⁶/ml

This informs us that if the laboratory reports a concentration result of 16 x 10⁶/ml then it will have a range of 16 x 10⁶/ml ± 1.4 (14.6 – 17.4 x 10⁶/ml).

Morphology – Uncertainty = 0.7%

This informs us that if the laboratory reports a morphology result of 4% then it will have a range of 4% ± 0.7 (3.3 – 4.7%).

Vitality – Uncertainty = 1.2%

This informs us that if the laboratory reports a vitality result of 54% then it will have a range of 54% ± 1.2 (52.8 – 55.2%).

Clinical impact of measurement of uncertainty will be communicated to users with annual website updates. Internal users of the service will be emailed directly (fertility and urology).

Clinical Impact of Measurement Uncertainty

Measurement uncertainty, either for one reported parameter or as a combination of multiple reported parameters could have a clinical impact. For example, if a concentration of 15 x 10⁶/ml is reported (oligozoospermia), given a UoM of 1.4 x 10⁶/ml, this sample could have a concentration of 16 x 10⁶/ml (normozoospermia).

For some users, a diagnosis of oligozoospermia, even at the borderline level of 15 x 10⁶/ml, could lead to the recommendation of an assisted reproductive technology such as intracytoplasmic sperm injection (ICSI) to promote the chance of fertilisation, when conventional IVF, or even natural conception, might be possible. 

We inform our users of this uncertainty and emphasize that the reports can be used to guide clinicians to determine how to proceed with further investigation and management of a subfertile couple. We stress that all reported parameters should be considered holistically.

Furthermore, as per WHO 2021 guidance, we inform users that many known and unknown female factors hamper the value of using only semen examination parameters to predict the prognosis for the couple of spontaneous or assisted fertilization.

The decision limits that we provide on our reports are derived from WHO 2021 and are derived from data characterizing the semen characteristics of a reference population (men whose partners had a time to pregnancy of 12 months or less). The data uses a diverse international populations, noting that the percentiles used do not represent distinct limits between fertile and subfertile men.

We highlight that a semen analysis is never prognostic of fertility, as it is the fertility potential of the couple that defines them as fertile or subfertile.

Unaccredited testing

The Department regularly reviews testing to reflect clinical service needs and advances in technology.  Consequently, some testing and equipment has not yet been reviewed at UKAS assessment visit.  The following testing and aspects of Cellular Pathology are not covered by UKAS 15189 accreditation:

Sputum Analysis - Differential cell count performed on induced and non-induced sputum samples. A developmental diagnostic tool to deliver accurate severe asthma diagnosis and phenotyping.  This will be a step towards patient tailored medicine delivery in severe asthma and will enhance our Trust position as a leading national centre in the management of severe asthma.

Digital Pathology - the Department is performing extensive clinical validation towards going live with digital pathology during 2024.  Hamamtsu S60 and S360 slide scanners are used as part of a Sectra IDS7 software solution for viewing and reporting digital images.  This will be subject to a UKAS 'Extention to accredited scope' application later in 2024.