I was attached to the Bone Marrow Lab for 3 weeks. Basically, the job of a medical/lab technologist is to get the aspirate and trephine sample (bone marrow biopsy) which the doctor extracted from the patient’s bone marrow and prepare the smears on the spot. Blood taken from the patient’s finger, pricked using a lancet is also smeared on the spot. The technologist is also responsible for letting the doctor know how much aspirate or trephine sample is needed and place it in the respective tubes and bottles. How much aspirate needed depends on the tests ordered by the doctor on the request forms.
After that, the slides are brought up to the lab where they are stained, dried, labeled, mounted and examined under the microscope. Patients’ information such as the name, hospital, ward, bed no., diagnosis, gender, age, etc. are recorded into the record book and also entered into the LIS.
During my 3 weeks there, I was able to observe bone marrow aspirations and also helped the doctor prepare the items needed for the aspiration when the nurse is busy with something else. I also helped my colleague place the aspirate and trephine sample into the respective tubes and bottles and ensured that all the required forms and tests requested by the doctor are ticked. After about 2 to 3 patients’ procedures are completed, I brought the slides up to the lab where I stained, labeled and mounted the slides and also recorded the required information into the record book and forms. During my free time, I practiced doing the various types of bone marrow smears except for the imprint smear as we do not have any extra trephine sample.
Preparation of smears
The 3 types of bone marrow smears to be prepared are:
1) Aspirate smears:
- Aspirate must contain bone marrow fragments.
- Same way as preparing a PBF smear.
- Accept only when marrow fragments are found mostly at the tail of the smear and free marrow cells can be seen in stained films.
2) Squashed smears:
- Few particles are placed on the centre of a clean slide.
- Another slide is used to gently compress to spread and disperse the particles as the slides are pulled apart.
3) Imprint smears:
- Bone marrow trephine biopsy sample is gently touched and rolled along between two clean slides.
The purpose of doing a bone marrow examination by preparing these 3 types of smears is to be able to show the different cells present in the patient’s bone marrow in order to diagnose certain conditions, assess the progress or stage of the patient’s disease and also to monitor treatment of certain conditions. Conditions include multiple myeloma, leukemia, lymphoma, anemia, amyloidosis, polycythemia vera, myelofibrosis, etc. Take for example leukemia, the patient could be suffering from acute or chronic myeloid or lymphoblastic leukemia and which type of ML or LL the patient is suffering from.
Staining of slides
1) The slides (2 aspirate, 1 squash, 1 imprint and 1 blood smear) are stained with Maygrundwald-Giemsa stain for the morphological classification of hematopoietic cells.
Maygrundwald-Giemsa stain
The Romanowsky stain consists of methylene blue and its oxidation products and eosin Y or eosin B. The combined action of these dyes produces the Romanowsky effect, yielding purple colouration to the nuclei of leucocytes and neutrophil granules and pinkish colour to the erythrocytes.
2) A separate slide (usually the aspirate smear) is stained with iron stain for the classification of anaemia associated with defective haemoglobin synthesis such as thalassemia, leukaemia, myelodysplasia and sideroblastic anaemias. The identification of ringed sideroblasts is used for the diagnosis of sideroblastic anaemia and myelodysplasia.
Iron stain
Cellular iron exists in the form of ferritin or hemosiderin (storage iron) in the bone marrow. Prussian blue iron stain is for the presence of hemosiderin (storage iron) in bone marrow and stainable iron in erythroblasts (sideroblasts) and/or erythrocytes. Hemosiderin stains blue-green; nuclei of cells stains red. Iron-containing granules will be demonstrated in siderocytes and normally in many of the normoblasts (precursor of erythrocytes) of human bone marrow. Extracellular iron deposits will be demonstrated in bone marrow. This siderotic material (hemosiderin) is distinct from ferritin, which is water-soluble and not detectable by Prussian blue reaction.
Examination under the microscope
Aspirate smears are able to show RBCs, WBCs and platelets. Squashed smears will be able to show whether the bone marrow is hypocellular or hypercellular. Imprint smears will be able to show cells that are present on the surface of the bone marrow core (bone).
Types of cells one will be able to see in the smears under the microscope: blasts such as myeloblasts, promyeloblasts, monoblasts and lymphoblasts, myelocytes, promyelocytes, promonocytes, eosinophils, basophils, erythrocytes, megakaryoblasts, megakaryocytes, prolymphocytes, etc.
Malerie Goh
TG02
15 comments:
Hi Malerie
What happens if your blood coagulates before you test it? Do you have to take it from the patient again?
Besides hypocellular or hypercellular cells, what other abnormalities can be observed?
Thank you
Ernest
0606330i
hello malerie
when collecting samples for the smears, you mentioned placing them in tubes and bottles. do the tubes and bottles have any form of anti-coagulant to prevent the samples from coagulating?
and since you have observed bone marrow aspirations, can you briefly describe how it is done?
thanks
rusydiana
tg02
Hey Malerie
What's the purpose of doing iron stain? Is it just to see the presence of iron? And if there's less iron than expected, what condition is it (iron-deficiency?)?
Hardina =)
Hi ernest,
Are u referring to the blood taken from pricking the patient’s finger? Firstly, when a patient is pricked, about 3 to 4 drops of blood is placed onto a slide. The smears are done immediately on the spot using a spreader. Very seldom the blood will coagulate before the smear is made. But if the blood coagulates before the smears are made, yup, we will have to prick the patient again.
As to what other abnormalities can be observed, do you mean cell abnormalities? Well, one example is blasts as mentioned in the post. Blasts are immature cells that are undifferentiated and should not be found in healthy individuals. Blasts include myeloblasts, monoblasts, megakaryoblasts, etc. If there is a presence of blasts in the patient’s blood/bone marrow smear, the patient may be suffering from leukemia. Further staining is then done to confirm what type of leukemia the patient is suffering from.
Malerie
TG02
hi rusydiana,
the samples collected include the aspirate and the trephine sample which are placed in the respective tubes and bottles and sent to the respective labs are for testing not for smearing. yup, the bottles and tubes does contain anti-coagulant. If the doctor orders for cytogenetics, the aspirate is placed in the tube containing lithium heparin. Other tubes include potassium-EDTA tube for molecular tests and EDTA tube for flow cytometry. Usually, the trephine sample is placed in a bottle containing formalin to act as a medium and sent to the histopathology lab for analysis.
There are 2 parts to the procedure. The first part is to get the aspirate. The second part is to get the trephine sample. First, iodine followed by septanol is used to clean the biopsy site. Local anaesthetic is administered to numb the site. Then, a special large-bore needle is inserted and rotated. A syringe is then attached to the needle and the aspirate is withdrawn. For the second part, the needle is inserted further inside. A marked probe is inserted to check the length of the sample and removed. An extraction cannula is inserted into the needle cannula. The needle is then rotated and the extraction cannula is removed from the needle. The marked probe is used to expel the trephine sample from the extraction cannula.
Malerie
TG02
Hi hardina,
Yup, the purpose of doing the iron stain is to test for the presence of iron – high or low and where the iron is deposited. As iron is needed in the production of RBCs, thus if there is low iron, there will be low production of RBCs, this is known as iron-deficiency anaemia.
Malerie
TG02
Hi Malerie. =)
Can I know if all 3 smears have to be done when a blood sample is collected? And is it possible to show pictures of the 3 different types of smears?
Thanks
Lyn
TG02
Hi Malerie,
Whats trephine? So you all collect the bone marrow using what kind of instruments?
Thanks!
LESLIE
Malerie~!
ask you have mentioned,
"Squashed smears:- Few particles are placed on the centre of a clean slide...."
what do you mean by "few particles"? what kind of particle is it?
Thanks,
YuMei, TG01
Hi Malarie,
What is myelodysplasia and what are the clinical symptoms and detection of this disease?
Thanks!
From: Benjamin Ma
Class: TG01
0606181F
i was just wondering, does the Maygrundwald-Giemsa stain preserve the cells in any way? I heard the Maygrundwald contains some sort of preservative right? If so, what does it contain?
-cornelyus
Hi Malarie!
I got questions for you. Really hope you could answer them for me.
1) You mentioned that you got to observe the doctor aspirate the bone marrow, was it a live demonstration? Could you briefly describe how it was done and the reagents used?
2) Just to confirm, when you mentioned bone marrow fragments, you actually mean bone marrow cells, right? Not the "bone" fragment?
3) This is a job-related question:
Just out of curiousity, in a hospital setting, the number of samples to process per day is a very large number, right? So, this require a tight time schedule and good skills to process the samples as fast as possible, right?
So, I would like to ask, how many samples do you get to process each day, and how much time is given to you spend to process per sample (may it be any kind of stainings)?
Many thanks
Quan Jun
TG02
Group 08
hello mal, u mentioned the different types of cells that can be seen under the microscope with those stains. so are there any cells that may be tehre but just that we cant see it and it may be impt too?
yuxuan
hi yuxuan,
basically those cells i've mentioned are the cells to look out for to differentiate what kind of leukemia the patient is sufferring from. most of them i've mentioned are abnormal cells.
take for example, normally smears are stained with those 2 stains and there is a presence of blasts present in the patient's PBF. the patient may be sufferring from leukaemia. but we are unsure wat kind of blasts are those. so to determine wat kind of leukaemia the patient is suffering from, a basic stain panel is used. this includes PAS, peroxidase n sudan black stain. PAS: not very useful for distinguishing AML from ALL. may stain positive for lymphocytes, granulocytes, monocytes and megakaryocytes. peroxidase is to confirm it's AML (look out for myeloblasts). sudan black is a marker to differentiate between AML and ALL. it is more sensitive in identifying myeloblasts. hope that answers ur question. =)
Malerie
TG02
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