Histopathology/haematology

Hello to all friends,

i was arranged to work in histopathology for the first 2 week, then 2 week in haematology. Now i'm in processing section.

But first let me tell you the Quality Assurance Programme “here”.

- Royal College of Pathologist Of Austrialasia
- External Quality Assurance Services (Bio-Rad)
- College of Pathologist Survery (general chemistry/immunology/transfusion medicine/Dignostic Virology/Haematology/General)

Unacceptable specimen criteria:- unlabeled specimen
- mislabeled/misidentified
- unidentified specimen
- specimen submitted in improper container
- specimens not accompanied by a cytology accession forms as appropriate
- incorrect/soiled accession form
- insufficient quantity of specimen
- empty container
- improper storage/ no clinical history

Note: the submitting physician/nursing station will be notified and will be expected to submit a fresh, labeled specimen. If not, the nurse need to personally identify the specimen.


First week: histology

1) In histo lab, documentation is very important. They must perform and maintain records which required by accreditation agencies (pre-analytical QC):

- run control slide on immunochemistry
- stain quality
- maintenance records/instrument maintenance
- calibration records
- records on type and number of tests done everyday, then evaluate every 6 months(draw bar graph & file)
- records of SOPs (not online. I understand some lab are online SOPs)
- duties of technologist
In short, they record down everything they do on paper and file it.

2) Screening and reporting of gynecological specimen- pathologist maintain work logs

3) Review of abnormal cases – identify areas for continuing education/seeking the opinion of outside consultant/documentation of all reviews is essential for quality assurance monitoring

4) Rescreening of negative cases

5) Cytology-histology correlation and clinical follow up- compare pre-malignant & malignant reports/helpful in directing further patient management

6) Retrospective reviews – IQA/efforts is made to minimize bias when reviewing cases

7) Measures of screening performance – correction & prevention measures

8) Proficiency testing and continuing medical education – EQA

9) Variability in practice - automation


Procedures from the start to the end
When the specimen arrives in the lab, the technologist will sign against the name of the patient, check whether the nurses send the correct specimens (should be in 10% formalin) or else they would call to the wards and check. Specimens are arranged to non-gynae/ gynae/ urgent.

Step2: tissue processing/ scribing
Tissues are either being cut/processed by technician(mostly like small specimen like lump/tonsil/EOS drop) or pathologist. Pathologist normally are in charge of handling bigger specimen which required specific orientation that the doctor want to observe( e.g. what is the degree of spreading of tumor cells in the breast).

Tissues are then transferred to cassette. Do u still remember the 12 steps processing? Let me remind you:

1 &2 steps: formalin
3step: 70% alcohol
4step: 95% alcohol
5&6&7steps: 100% absolute alcohol
8&9&10steps: xylene
11&12steps: hot liquid paraffin wax

After the tissue are embedded into metal mould and sectioned. Some of the hospital lab have different method in sectioning. My lab is as following steps:

1) place the cell blocks onto the block holder clamp. Ensure that the block is perpendicular to the microtome blade.
2) A blunt knife is used to shave off the excess wax from the block to expose the specimen. If the t/s containing unsuspected mineral deposits, the block are placed into decalcifying solution for surface decalcification. If the specimen is skin, the block are placed into softener (ya! the one we used to wash our clothes with—make it softer)
3) Change the blade to new sharp blade
4) Transfer the blocks to a cold plate
5) Refit the block to the clamp
6) Sectioned the block @ 4-5 microns by rotating the microtome handle in the clockwise/anti-clockwise (depends on the machine) to produce thinner sections. The number of microns used also depend on the pathologist.
7) Lift the sections onto the alcoholic water to spread the sections (unfold)
8) Transfer the sections onto the warm water bath (45 degree) and use a clean glass slide to “fish” out the sections


These slides are then arranged into a rack (facing one direction) and placed into an oven @ 75 degree for 10mins.

When the time is up, the rack are placed into a containing container of the auto-stainer. (staining methods will be explained later)

Interpretation: histological slides are examined under a microscope by a pathologist and then he/she will report his/her findings. Pathologist will compare results among themselves to give a best interpretation.

H&E staining is a very common staining procedures. I am sure that everyone should know so I’m not going through it. Beside H&E stain, special stains is another methods to identify glucogen/carbohydrates/minerals.

Most common special stains:
- periodic Acid Schiff’s (PAS)
- Periodic Acid Schiff Diastase
- Alcian blue
- Congo red
- For other pls check this website: www.hoslink.com


Frozen lab is normally located next to a operating room. Pathologist will examine the t/s while the surgery is still taking place.

Reasons:
1) If a tumor appears to have metastasized, the pathologist and the surgeon will decide whether there is any point in continuing the operation.
2) If a tumor has been resected but it is unclear whether the surgical margin is free of tumor, an intraoperative consultation is requested to asses the need to make a further resection for clear margins.
3) In a sentinel node procedure, a sentinel node containing tumor t/s prompts a further lymph node dissection, while a benign node will be avoided.
4) Rapid examination of a lesion might help to identify the possible cause of a patient symptoms.

Steps:
1) When the specimen arrives, it will be immediately be assigned with a number which will be entered on the examination forms, and histology logbook and the time of receipt recorded.
2) A dignosis should be made and called to the operating room in 20mins
3) The pathologist will check the specimens, dissect if necessary, and will place the t/s to be sectioned onto a bed of OCT compound on a crystat chuck. Liquid nitrogen is used to froze the chuck.
4) The specimen is then inserted into the holder arm of the cryostat, and t/s sectioned @ 5µm
5) Once the section has been cut, it can be picked up by a glass slide over it (the t/s will “melt” onto the slide due to different in temperature). Slides are labeled with accession number.
6) 1 slide for H&E staining. Another for Tol Blue staining
7) Pathologist will diagnosis after observing the slide and record on the logbook and submission sheet. Time of receipt is made.
8) Patient’s identification checked and confirmed before delivery of any verbal report to the surgeon.
9) The remaining of t/s will undergo normal histopathology
10) Report records permeantly onto patient’s records

Second week: haematology

D-Dimer test

1) Introduction

D-dimer is a protein that is released into the the circulation during the process of fibrin clot breakdown. D-dimer represents an area of cross-linked fibrin degradation product that originated from the breaking down of the fibrin clot network during the body’s repair mechanisms. These fragments are released from the clot by the action of the enzyme plasmin. D-dimer present in circulation is used as an indication of a blood clot being formed and broken somewhere in the body.

Blood Activation


Thus it is possible to use D-dimer as a test for many medical conditions and complication. In normal people, there is a very low background level of D-dimer. Abnormal levels (elevated levels) of D-dimer can be formed in patients with Deep Vein Thrombosis (DVT), pulmonary Embolism (PE) and disseminated Intravascular Coagulation (DIC).

2) Principle

The latex particles provided in the D-D1® Test are coated with mouse anti-human D-dimer monoclonal antibodies. Test samples containing D-dimers when mixed with the latex particles suspension make the particles agglutinate. At a predetermined concentration of D-dimers that the D-D1® Test is designed for, the agglutination of the latex particles produces macroscopic clumps that can visualized by the naked eye.

3) Specimen

Blood in sodium citrated tube are centrifuge for 3 mins @ 1500rpm. Plasma is needed


4) Proceduce



The 5&6week: Processing section

I heard from the rest that different hospitals have different way of processing the specimen. For example, pei shan’s lab processing section is automated while mine is manual check.

In my lab, the processing section is where the patients specimen from various wards, clinic, A/E and other departments will reach the lab via pneumatic system. This is also the place where all the datas of patients and tests ordered are keyed into the computer before dispatching the specimens to the various sections where the tests are performed. Thus Processing is the most important section among all sections.

Let me explain the proceduce in details

Firstly, the canister carrying specimens will travel from the wards, clinic, A/E and other department through the pneumatic system to the lab. The lab have three automated lines to receive and send out results. It also have three manual lines to send results or rejected out the code of the desired wards. E.g. code 3300 is for ward 33

The specimens will come with their requested forms, and these forms are to be sorted into awaiting/urgent and non-waiting. Time has to be zapped on awaiting/urgent/A&E/blood gas/seminal analysis/blood bank forms/ add-in tests. Priority is given to the above forms.

In addition, forms for bacteriological test, histopathological tests and external tests have to be placed at certain in-tray as they will not be run in the general lab. These tests are only available in other labs in SGH/KKH/TTSH.

Next I learned to do pasting of labels onto the tubes. The most common labels are biochem, cell dyn, ESR, Hba1c, blood gas, RA, INR, Axsym, TIBC, immuno, Stool. The tubes come in different colour too( pink cap-EDTA Blue-sodium citrate Grey-fluoride Dark Blue-zinc Yellow-plain).

Labeling are important because it ensure that the samples are the correct patients and the test ordered must match. Plain tube are centrifuged at 3500rpm for 5 mins while the rest are sent to other sections. Rejection part is e same as histo(refer back to the top)


ps: sorry if it's too long

cheers,
elaine