I'm now attached to the Bone Marrow Morphology Lab for 3 weeks. This lab has the most senior medical technologists and is considered one of the most exciting and challenging labs amongst all the others labs in Haematology. When situated in this lab, one will have the golden opportunity to go down to the wards and experience how the bone marrow is being aspirated out from patients! Indeed it may give one a nauseating feeling, but this is what makes the job in this lab so exciting about. ;)
Bone Marrow Preparation, Staining & Reporting
After clinical history, physical examination and the review of peripheral blood film, bone marrow examination is the next most important step used in the diagnosis of haematological disorders. It is the only way used in the classification of leukaemias and myelodysplasias. It allows the assessment of the body iron stores, marrow cellularity, myeloid to erythroid ratio, maturation of cell lines and relative quantitation of eosinophils, lymphocytes and plasma cells. In addition, infiltration by fibrosis and other malignant cells can also be diagnosed. It is also used to assess response to treatment in leukaemias and to assess prognosis in aplastic anaemia and agranulocytosis. The absence of iron stores help to differentiate iron deficiency anaemia from other hypochromic anaemias. Trephine (a fixative for the bone) biopsy may help to diagnose myelofibrosis, aplasitc anaemia, malignant lymphoma or secondary carcinoma.
Equipments & Materials
1. Microscope
2. Multitimer
3. Differential counter
4. Glass slides
5. Spreader
6. May-Grunwald stain
7. Giemsa stain
8. Buffered distilled water pH8.6
9. Mounting medium DPX
10. Adhesive labels
11. Reporting worksheets
Specimen Collection
The marrow aspirate can be obtained from the posterior superior iliac spine due to its accessibility and relative safety which a sample can be obtained.
Preparation of bone marrow smears
Bone marrow smears can be made directly at the beside during aspiration procedure. The sample is then sent to the lab for slide preparation.
Wedge smears
About 0.3ml of bone marrow from the first stringe is put onto a clean glass slide to allow the blood to drain away onto another slide. The marrow fragments tend to adhere to the slide and most of them will be left behind. A smooth edge spreader of not more than 2cm in width is used to make 10-12 wedge smears of size 2x3cm. The marrow fragments are then dragged behind the spreader and they leave a trail of cells behind them. Differential counts are then performed by the senior staff.
Squashed preparation
A few particles are placed on the centre of a clean glass slide in which another slide is gently compressed to spread and disperse the particles as the slides are pulled apart.
Trephine imprint
Several touch or imprint of the bone fragment are prepared by gently touching the core and rolling between 2 slides.
Handling conditions
1. All bone marrow smears are labeled with the patients' initials immediately after preparation. They are kept in separate trays and brought back to the lab with request forms to be filled in by staff.
2. A unique marrow number is assigned to each patient sample and are written in request forms, record book etc.
3. 2 wedge smears, 1 blood film, 1 squashed preparation and an imprint are selected for May Grunwald Giemsa staining. Another wedge smear with visible particles is used for iron staining. Controls with normal/increased iron stores are stained in parallel with the patients' smear.
Bone Marrow Smear - May Giemsa Stain (x1000)
Image from: pathy.med.nagoya-u.ac.jp/atlas/doc/node76.html
4. A set of buff cards and adhesive labels for storing the smears together with a worksheet with patient's details are prepared.
5. Any unstained smears are kept in closed containers for 2 weeks for further tests, if necessary.
I've only spent 2 days in this lab, so this is whatever I've observed and learnt so far! If you guys have any queries, please do not hesitate to ask. ;)
Signing off,
Felicia
0703345I
Hey Fel,
ReplyDeleteAre there any other storage conditions that we have to keep in mind when we keep the unstained smears for further tests?
Yvonee
0703189A
Felicia,
ReplyDeleteAre there other fixatives used besides Trephine i.e. used for bone biopsy? What are the differences between Trephine and other fixatives? Can you list me examples? Thank you.
Li Yinliang Alex
TG02 0704894E
Group 8
19 August 2009
Hey Felicia,
ReplyDeleteHow long are the slide kept and how long does the slide last with and without mounting.
Cheers
Tiong HAn
Exactly how is the bone marrow aspirate perform? Just by injecting a needle into the spine and suck? How fine is the needle?
ReplyDeleteIs it a must to use all 3 types of bone marrow smears? If no, which will be the most preferred smear to perform?
Alvin
Hey Yvonee,
ReplyDeleteFrom what I've seen so far, the unstained bone marrow smears are kept in a slide box and the box is then placed in room temperature in the lab. No other specific conditions are required.
Alvin,
The bone marrow aspiration is usually done before the bone marrow biopsy. For the aspiration, a hollow needle is inserted through an incision and on through the bone and into the bone marrow cavity of your iliac crest. Because local anesthetics aren't able to numb the interior of your bone, you may feel a deep, aching pain when the needle is fully inserted.
A syringe is used to draw a sample of the liquid portion of the bone marrow into the hollow needle. As the liquid is drawn up, you may feel a painful stinging, sucking or pulling sensation, which may travel down your leg. The aspiration takes only a few minutes. You may need to have several samples taken.
And yes, it is important to perform all 3 smears so as to microscopically examine the bone fragments.
Stock needles are made with a special extra heavy 12 gauge needle stock (3.0 mm external diameter with 1.5 mm internal diameter). This gives the needle great rigidity for ease of penetration.
Alex,
So far, trephine is the only fixative that they use to fix the bone. As for other types of fixatives, I did some research and here's what I've found;
Other fixatives, such as Bouin's fixative and the mercury-based fixatives, Zenker's and B5, are also used with trephine biopsy specimens. These fixatives give excellent preservation of cytological detail but are less practical in labs processing a wide range of tissues.
If Zenker's fixative is used, the reactivity of Ab used for immunohistochemical staining may be affected as Zenker's fixative can destroy chloroacetate esterase (a stain to demonstrate the presence of granulocytes) activity.
If B5 fixative is used, the duration of fixation is more critical. If it lasts for for than 6h, the bones will harden and it would be difficult to cut the sections for examination.
Tiong Han,
The slides are kept for a week, be it mounted or not mounted.
Hope I am able to answer to your questions!
Fel
Hi Felicia.
ReplyDeleteWhat happens to the unstained samples after 2 weeks? What are the changes or effects on the specimens chemically/microscopically that makes it unable to be used after 2 weeks?
Thanks!
Siti Hawa
tg02
group 10
0704087F
Hey Siti,
ReplyDeleteThe unstained samples will be disposed as there will be slight morphological changes in the slides. For example, the RBCs might clump together, or certain red cell indices might vary. Dust particles might be trapped in between the slides, causing them to adhere as precipitates. Thus, it might be difficult to view the cells microscopically.
Hope that answers to your question! :)
Fel
Hi Felicia,
ReplyDeleteHow much of bone marow aspirate do you need to all the testings and prepare the unstained slides? And will there be any chances that bone marrow aspiration is required to be done again if the specimen is insufficient or you need to repeat the testings after 2 weeks?
Thanks =)
Lok Pui
Hey Lok Pui,
ReplyDeleteAn aspiration usually yields approximately 300 μL of bone marrow. Any volume greater than 300 μL is not recommended as it may dilute the sample with peripheral blood.
Unfortunately a second bone marrow aspiration must be done if there is not enough marrow for further testing. This is however not common as 300uL of marrow is sufficient for all required testing to be carried out. ;)
Fel