Although conventional cytogenetics analysis is one of the essential tools for diagnostic investigation, there are several limitations too. By knowing the limitations of this study, we can find out the alternative to overcome it. Thus, I have listed down some of the limitations below.

Limitations of Conventional Cytogenetics Analysis

1. Cannot Assess Non-dividing Cells

By using conventional cytogenetics technique, we can only assess the dividing cells. This limitation becomes obvious when we wish to analyze the diseases like chronic lymphocytic leukemia, malignant myeloma and the solid tumors. Yet, the FISH (Fluorescence in situ hybridization) technique and molecular analyses can be used to assess the non-dividing cells if the specific abnormality is known.

2. Low Efficiency

As we all know, the cytogenetics test has to be handled by the lab technologists step by step. The lacking of automation in sample processing causes the process becomes time consuming. Furthermore, only few of the cell divisions are analyzed. However, FISH and molecular analysis can be used to screen more than hundreds cells at once.

3. Insufficient Result

Sometimes, if there is insufficient cell to be analyzed, or only normal cells are found, the result is not useful for the patients.  Therefore, no matter how hard it is, the cytogeneticists have to try their best to find out the abnormalities so that the useful result can be provided to the patient.

4. Insignificant Abnormality Might be Found

It is the cytogeneticists’ responsibility to determine the clinical significance of the abnormality that was found. Sometimes, the abnormality found is not significance.

5. Poor Morphology

Sometimes, it is difficult for the cytogeneticists to detect the abnormality as the chromosome morphology is poor. This becomes obvious for detecting the Acute Lymphoblastic Leukemia clones as the chromosome morphology for those cells tend to be very poor. Luckily, we can still use the FISH to make sure the abnormality accurately.

Summing up, the limitations of the conventional cytogenetics analysis can be overcome by using FISH technique and other molecular techniques. Yet, the cytogeneticists have to put more effort to lessen the limitations so that the best result can be presented to the patients. Thanks for visiting Cytogenetics Cancer Research blog!

(Reference: Cancer Cytogenetics edited by John Swansbury)

5 Limitations of Conventional Cytogenetics Analysis is a post from: Cytogenetics and Cancer Research

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Conventional Cytogenetics Analysis is an important tool in medical field. There are many applications of conventional cytogenetics analysis. It is good to know the strengths of this analysis technique so that we can know the most appropriate genetic assays to be used.

Generally, a clinician may ask for the conventional cytogenetics analysis even though it may not be appropriate for the information sought. Therefore, it is essential for the clinician to know the application of this analysis. On the other hand, the cytogeneticist may be consulted to use the best analysis for the patients.

Applications of Conventional Cytogenetics Analysis

1. Detect the Presence of Abnormal Cancer Cell

We can easily detect the abnormal clone by using karyotyping. It is helpful in differentiating between reactive conditions and malignancy if there is an abnormal clone present. For instance, detecting an abnormal clone is good when we are investigating a pleural effusion, anemia or lymphocytosis. Yet, there might more than one abnormality present in the same sample too. Sometimes, most of the analyzed cells are normal. But it does not mean that there is no abnormality. It might because of the abnormal cells were not grown well during the culture process.

2. Confirm the Diagnosis

The conventional cytogenetics analysis is useful to clarify the diagnosis. For instance, the clinician can only diagnose for the Down syndrome according to the physical appearance of the patient. We can use conventional cytogenetics analysis to help to confirm the diagnosis.

3. Indicate the Prognosis

Cytogenetics analysis is one of the most powerful prognostic indicators in hematologic malignancies. This effect persists despite advances in treatment. It is important for solid tumors prognosis too. Therefore, sometimes the cytogeneticists feel hesitate to report the karyotype as normal. The failure of finding a cancer clone implies only the normal cells were analyzed or the genetic abnormality was undetectable.

4. Help in Choosing the Best Treatment

The clinician will always take into account the cytogenetics analysis when choosing the treatment for the patients. For example, a bone marrow transplant will only be used when the risk of dying from the malignancy is relatively higher than the risk of doing the transplant according to the cytogenetics analysis. Besides, this analysis can assist the clinician to tailor the treatment according to the needs of the patient so that the risk of relapse against the risk of therapy-related death can be balanced.

5. Keep Tracking the Response of Treatment

Conventional cytogenetics analysis is good for monitoring the response of the treatment to the patient. For example, we can use this analysis to monitor the effectiveness of the bone marrow transplant to the patient.

6. Support Further Research

By using the result of conventional cytogenetics analysis, we can do the further research for particular abnormality. We can publish the usual findings as case reports to provide more information to the public. It will become useful when the same abnormality is discovered in other patients.

In conclusion, the cytogenetics study is still playing an important role for diagnostic investigations of patient who has hematologic malignancy or solid tumors. Yet, this can be assisted by other genetic assays too such as microarray. Personally, I really appreciate the applications of cytogenetics analysis. I will explore more about it so that I can really use it to help in increasing the accuracy of diagnosis and prognosis. Keep on reading more about cancer cytogenetics in Cytogenetics Cancer Research blog.

(Reference: Cancer Cytogenetics edited by John Swansbury)

6 Applications of Conventional Cytogenetics Analysis is a post from: Cytogenetics and Cancer Research

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After coming back from Singapore, I have been given a precious bone marrow sample to do the cytogenetic test from Prof. Hany to be tested. This was the first time for me to deal with the bone marrow sample. With excitement, I started off with the culturing process.

Obstacles of Cytogenetic Test

However, there were many obstacles for me to do the test. The materials and reagents that I need for doing this test are not available at that time, especially for the essential culture medium. Consequently, I need to think of the alternative. I tried to use the transfer medium provided by National University Hospital Singapore to do the culturing process with the hope to grow at least some leukemic cells from the sample.

Furthermore, I do not have the pre-made ethidium bromide solution yet. The ethidium bromide solution is important to elongate the chromosomes. So, I have to make it myself by using the powder-formed ethidium bromide.

Outcome of Cytogenetic Test

After culturing and harvesting the bone marrow cells, I used the cell suspension obtained to prepare the slide. Then, I viewed the slide under the microscope. There were just few metaphases and the chromosomes not spread well. I am going to stain the chromosome on the next day. I hope that I can still get some good metaphases to be analyzed via imaging system.

Troubleshooting the Problems

In my opinion, this is not a good result that I want to get from the cytogenetic test. Why?

1. There were a lot of dead cells when I viewed the slides under microscope. This might probably because of the cells not grow well during the culturing process.

2. The length of the chromosomes was short. This indicated that the elongation step was not performed well.

3. The mitotic index was low. This might because of the metaphase arrest step was not performed well. Yet, the mitotic index is closely related to the condition of the sample and the patients too.

4. The chromosomes not spread well. This might because of the slide preparation step was not performed well. This step closely related to the technique used by the technologist. I think I am not doing well when performing this step.

Solutions for the Problems

To solve those problems, there are many optimization steps that I need to carry out.

1. I have to use the appropriate culture medium in order to make sure the cells can grow well especially for the leukemic cells. I am ordering the Chang Medium for bone marrow culture from Irvine Company.

2. The home-made ethidium bromide solution seems like not work so well. Therefore, I decided to order the pre-made ethidium bromide solution too to make sure the chromosomes can be elongated.

3. The amount of the reagents used should be accurate.

4. I will try my best to improve my technique when performing the slide preparation step to make sure the chromosomes can spread well on the slide.

Summing up, after the first trial, there are still plenty of things for me to do and learn. Tomorrow I will continue with the staining step and start to analyze the metaphases. Stay tuned in Cytogenetics Cancer Research blog to see what I will get from this first trial.

First Trial of Cytogenetic Test on Bone Marrow Sample is a post from: Cytogenetics and Cancer Research

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Cytogenetic analysis is a powerful technique for bone marrow diagnosis. This is the technique that I wish to use for my leukemia research. For your information, I just went to National University Hospital of Singapore (NUH) to learn the cytogenetic analysis for bone marrow diagnosis last week week. Before I go there, I need to know a little bit about this technique first. Therefore, today we will explore about this technique.

In hematological neoplasms, the specific and non-random chromosomal abnormalies are commonly found. Those abnormalities become a main factor in pathogenesis. There are some of the hematological neoplasms are defined according to the presence of specific chromosomal abnormalities rather than referring to hematological or histological features. We can see an example where a specific subtype of AML which is M4E_o/inv(16)(p13q22) /CBFβ-MYH11 fusion AML is better defined by the presence of inversion of chromosome 16. Sometimes, the presence of certain chromosomal abnormalities also provides prognostic information too. Furthermore, cytogenetic analysis is good in distinguishing a neoplastic from a reactive process. For instance, the demonstration of clonal cytogenetic abnormality proves that idiopathic hypereosinophilic syndrome is actually eosinophilic leukemia.

Protocol of Cytogenetic Analysis

As we all know, classical cytogenetic analysis can be performed only on cell suspensions which are obtained from peripheral blood or bone marrow. For hematological neoplasms, the metaphase spreads will be examined. Those metaphase spreads must be prepared from blood or bone marrow aspirates either with direct preparations or cell culture. The cells will be arrested in metaphase stage by using colcemid. After the harvesting process and dropping process, the chromosomes will be stained by using Giemsa or quinacrine mustard. With the visualization of the chromosomes, all the individual chromosomes will be classified according to their size, position of the centromere and also the banding pattern.

There are two ways that the finding can be illustrated. The findings can either be illustrated by a karyogram, which is an ordered array of chromosomes or illustrated by karyotype.

In conclusion, the protocol of processing bone marrow specimens for cytogenetic analysis is almost similar to the conventional cytogenetic test. In my next post, we will talk about the applications of cytogenetic analysis for bone marrow diagnosis and its limitations. Stay tuned in Cytogenetics Cancer Research blog!

(Reference: Bone Marrow Pathology written by Barbara J. B., David M. C., Irvin A. L. and Bridget S. W.)

Cytogenetic Analysis for Bone Marrow Diagnosis is a post from: Cytogenetics and Cancer Research

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After the second day of my training in National University Hospital (NUH) Singapore, I started to try to find the fastest ways to improve myself in cancer cytogenetics. As I mentioned in my previous post, I found that there are five deficiencies for me in doing leukemia cytogenetics. Therefore, I have to try my best to turn the things around.

Learning While Doing the Research

Yes, I have to admit that I do not have adequate knowledge about cancer cytogenetics. However, I can learn the knowledge while doing my leukemia cancer research. Thus, I planned to read through the reading materials about the leukemia first especially for the acute lymphoblastic leukemia (ALL). This will be the beginning step for me now.

Fully-utilized the Equipments

As it is almost impossible for me to get the new machines and equipments that are required for cancer cytogenetics, I will try my best to modify the protocols so that it may work well with the machine and equipments that we have currently. I will look through the whole University of Malaya Medical Centre (UMMC) to check the availability of the machines and equipments in other laboratories.

Analyzing Skill

In order to improve my analyzing skill, there are three ‘more’ that I have to achieve.

i. View More

I have to view more metaphases especially from the cancer patients so that I can familiar with the characteristics and morphologies of the abnormal cells.

ii. Try More

I have to try to karyotype and analyze more metaphases even though I might make a lot of mistakes. I will apply the try and errors method to make myself good in analyzing.

iii. Consult More

During the time I do my cancer cytogenetics research, I might need a lot of guidance from an experienced Cytogeneticist. So, I will consult more people by using internet discussion group, email and so on.

Accumulate my Experience

Although I need some times to accumulate my experience in cancer cytogenetics, I can consult the experienced staffs in my laboratory. I think they are willing to teach me as they are friendly, helpful and kind.

Spend my Time Wisely

Yes, another main problem for me is the time. I need to race with the time. Anyway, what I can do is just try my best and do the best. I have to more focus to cancer cytogenetics now. Then how about blogging? I might not update the blog as frequent as now start from today. I have to do so. I am so sorry about this. But I will not just dump this blog away. I love this blog very much. How about you? Haha…

Summing up, there are a lot of things that I need to do start from now so that I can catch up the pace for cancer cytogenetics. Before I end up this blog post, I would like to thank to all the members of NUH Cytogenetics Lab. They have taught me a lot of things. Thanks to Prof Hany too!

5 Ways to Improve Myself in Cancer Cytogenetics is a post from: Cytogenetics and Cancer Research

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After the first day of my training in National University Hospital (NUH) Singapore, I have found out that there are five of my deficiencies in doing leukemia cytogenetics.

1. Basic Knowledge

Firstly, I am lacking of basic knowledge about leukemia as well as cytogenetics. When listening to the explanation of one of the staffs in NUH, I found out that I cannot fully understand what she was talking about. There are a lot of things that I never heard before.

2. Analysis Skill

The second deficiency is I still do not have the ability to analyze the chromosome yet. As what you know, we have to analyze the result after we have done the cytogenetics test. However, I have not mastered the analyzing skill yet. Without the analyzing skill, I could not identify the chromosomal abnormality even though I manage to get a good result.

3. Equipments and Materials

Besides, not all the equipments and materials that are necessary for the bone marrow cytogenetics analysis are available in my laboratory in University of Malaya Medical Centre (UMMC). I have to spend a lot of money to buy the new equipments and materials for doing the leukemia cytogenetics. Most of these equipments and materials are very expensive.

4. Experience

This is my major deficiency in doing leukemia cytogenetics. I am lacking of experience. For doing leukemia cytogenetics, experience is essential. Sometimes, it depends to our experience when analyzing the chromosomes. Without the experience, it is too difficult for me to do everything alone without any guidance.

5. Time

Last but not least, I am lacking of time now. I just have another 4 days time to learn everything in NUH. After that, I have to start the research on bone marrow cytogenetics analysis in UMMC. Is it an impossible task for me? I hope that I can go through it smoothly.

Thus, there are a lot of things that I need to do to improve myself. I have been given some reading materials about the hematology malignancy. I have to read through it as soon as possible. That’s all for today blog post. Thanks for visiting Cytogenetics Cancer Research blog!

5 of My Deficiencies in Doing Leukemia Cytogenetics is a post from: Cytogenetics and Cancer Research

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After taking about an hour flight, finally I am able to be in Singapore and waiting for the training in National University Hospital (NUH) Singapore. I think this will be an exciting and challenging journey for me. It is because I have to learn everything especially about the leukemia cytogenetics technique within one week time!

Get Ready for the Leukemia Cytogenetics Training

Since arriving Changi Airport, I have to adapt to this new environment so that I can feel comfortable to learn something here. After settling myself in a nearby hotel, I started to plan and think for my training. What am I going to learn for this training?

i. Objectives and Implications

First of all, I wish to find out the objectives and implications of doing the leukemia cytogenetics. As far as I know, the researcher used cytogenetics analysis to look for the chromosomal abnormalities that occurred for the leukemia patients. The study serves two objectives. The first one is to increase the accuracy of leukemia diagnosis as well as providing prognostic information and allowing the more rational selection of therapy for a particular patient. The second reason is to detect the sites of consistent rearrangements so that we can know the precise localization required for the isolation and cloning of DNA from these regions. This is important for the further investigation of leukemia disease. There will be more objectives and implications that I wish to know from this training.

ii. Materials and Methods

In my opinion, this is the main purpose for me to come for this training. I wish to learn the methods and the materials that required for leukemia cytogenetics. The chance will be given for me to see step by step how the scientists in NUH doing the cytogenetics analysis for leukemia patients. For your information, the scientists in NUH have done a great job in cytogenetics analysis for leukemia. Their skills are awesome! I am sure that I can learn a lot of applicable skills from them.

iii. Knowledge

Next, I wish to gain more knowledge about cytogenetics analysis and leukemia from this training. Before this, I can only read through the related books and learn some theories from it. Theories are not enough in cancer research. However, start from tomorrow, I can absorb more applicable knowledge by asking a lot of questions to the scientists here. Their experience and professional skills will be my precious treasure to be hunt during the time in Singapore.

iv. Troubleshooting

Besides, I hope that I can get the ideas on how to troubleshoot the problems that might occurred during the cytogenetics analysis. This is essential for me as I will do the whole process of cytogenetics analysis alone after this one week in Singapore. If I was unable to troubleshoot the problems, it is not possible for me to know how to modify or optimize my skills or techniques when I cannot get the result. As we all know, there are plenty of factors that might influence our experiment results. The condition and environment in NUH might not exactly the same with University of Malaya Medical Centre. Thus, I have to prepare myself for that.

v. Attitude

Another thing that I wish to highlight is the attitude. In my opinion, the attitude can make things different. We can copy the same methods, but without the right attitude, we might achieve nothing. For this reason, I will observe the professionalism and attitude of the scientists in NUH as example to follow.

Lastly, there are five days for me to learn everything from here. I hope that I can be humble and enthusiastic in the whole learning process. Thanks to Professor Hany Ariffin for arranging all of these for me! Thanks to UMCRI to support me for this training! I will grab this opportunity to improve myself.

5 Things I Wish to Learn in Singapore for Leukemia Cytogenetics is a post from: Cytogenetics and Cancer Research

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Bone marrow specimens can be examined cytologically and histologically. There are many types of bone marrow examination. I have talked about bone marrow aspiration and trephine biopsy, the two main bone marrow examinations previously. Today, we will discuss about the advantages and complications of bone marrow aspiration and trephine biopsy.

Advantages of Bone Marrow Aspiration

Bone marrow aspirates allow a wider range of cytochemical stains and immunological markers if compare with histological sections. It is good and can be performed well alone for investigating patients with suspected iron deficiency anemia, anemia of chronic disease, megaloblastic anemia, acute leukemia and so on. However, bone marrow aspirates are not equal for demonstration of fine cytological detail.

Advantages of Trephine Biopsy

One of the important features for process of trephine biopsy for diagnosis is when a dry tap or blood tap occurs because of the marrow being fibrotic or densely cellular. Besides, only trephine biopsy permits a complete assessment of bone marrow architecture and the pattern of abnormal infiltrate distribution. It performs well in investigating suspected aplastic or hypoplastic anemia, lymphoma, metastatic carcinoma, myeloproliferative disorders and bone diseases. Furthermore, trephine biopsy is more useful in diagnosing patients with the advanced stages of HIV infection in whom hypocellular if compare with bone marrow aspiration. Yet, trephine biopsy causes more pain than bone marrow aspiration to the patient.

Complications of Bone Marrow Aspiration and Trephine Biopsy

There are many complications of bone marrow aspiration and trephine biopsy although very rare. Cardiac and great vessel laceration are two examples of complications causing by bone marrow aspiration and trephine biopsy. Another example is hemorrhage (bleeding). If hemorrhage occurs, a prolonged firm pressure is necessary after the bone marrow aspiration or trephine biopsy to ensure that bleeding has stopped. If trephine biopsy is worked on bone with an abnormal vasculature, hemorrhage will be the problem. Some of the osteoporosis patients have severe retroperitoneal hemorrhage after bone marrow aspiration and trephine biopsy have been carried out. Sometimes, the lateral cutaneous nerve of the thigh will be damages due to the poor skill. Pneumothoraces can be observed after sterna aspiration and sternomanubrial separation too.

In conclusion, both of these techniques have advantages and limitations respectively. Thus, we can use both techniques as complementary to get the best specimens examination. The complications of these two techniques have to be prevented so that there will be no problem for the patients during examination. If you wish to know more about bone marrow in Cytogenetics Cancer Research blog, you may follow the bone marrow category.

(Reference: Bone Marrow Pathology written by Barbara J. B., David M. C., Irvin A. L. and Bridget S. W.)

Advantages and Complications of Bone Marrow Aspiration and Trephine Biopsy is a post from: Cytogenetics and Cancer Research

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Trephine biopsy is one of the essential techniques for bone marrow examination. The process of trephine biopsy is not difficult to be done. However, the two principal strategies of fixed trephine biopsy specimen preparation have pros and cons too.

Sometimes, the difficulty of cutting tissue that composed of hard and soft but easily torn bone marrow will be the problems of trephine biopsy. To solve this problem, the specimen is decalcified or embedded it in a substance that can harden the bone marrow. For decalcification, the weak organic acids such as formic acid and acetic acid can be used. On the other hand, the chelation with ethylene diamine tetra-acetic acid (EDTA) also can be applied to decalcify the specimen.

There are many consequences of decalcification and paraffin-embedding of specimen.

i. There will be the shrinkage and loss of cellular detail as the sections become thicker.

ii. The nuclear staining may turn into blur.

iii. Some of the cytochemical activities will be lost. For instance, the chloro-acetate esterase activity will be lost after decalcification of bone marrow with weak organic acid is done.

On the other hand, plastic-embedding techniques are relatively more expensive than paraffin-embedding techniques. Moreover, the technical steps of plastic-embedding techniques are more difficult. Yet, there are many advantages of using this method. Unlike paraffin-embedding techniques, there will be no shrinkage, no loss of cellular detail as well as thinner sections of plastic-embedding techniques. This means that a finer cytological detail can be easily obtained by using plastic-embedding techniques. Furthermore, some of the enzymatic activities can be retained. Although immunological techniques are more applicable to paraffin-embedded than plastic-embedded specimens, excessive background staining is a common problem.

There are various qualities of plastics that can be used for embedding. One of it is methyl methacrylate. This kind of plastic needs a long process and it is not suitable for routine diagnostic laboratories. Another example is glycol methacrylate. Glycol methacrylate is a better choice for plastic embedding techniques.

In conclusion, the process of trephine biopsy is simple but still needs a lot of optimization approaches. We have to be cautious when dealing with the specimens. We have to remember that the sample is very precious. We cannot afford to lose it just because of our careless. Thus, try to do the best!  Stay tuned in Cytogenetics Cancer Research blog.

(Reference: Bone Marrow Pathology written by Barbara J. B., David M. C., Irvin A. L. and Bridget S. W.)

Process of Trephine Biopsy is a post from: Cytogenetics and Cancer Research

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Trephine biopsy is another popular technique of bone marrow examination. Trephine biopsy can be called as needle biopsy too. In my previous post, we have talked about bone marrow aspiration which is another famous bone marrow examination technique. Today, we will explore deeply for trephine biopsy.

Location of Trephine Biopsy

Trephine biopsy is usually done on the iliac crest no matter posteriorly or anteriorly. Generally, most of the people prefer posterior technique than anterior. As we mentioned in the previous post, if the bone marrow aspiration and trephine biopsy are carried out at the same time, they can be done through the same skin incision with the two areas of periosteum that being infiltrated with local anaesthetic and with the needle that being angled in various directions.

Use of Bone Marrow Specimens by Trephine Biopsy

Trephine biopsy can produce the specimens that are suitable for histological sections, touch preparations or imprints, and electron microscopy. Similar to the specimens yielded by aspiration, the specimens of trephine biopsy can be used for cytogenetic test too. Yet, the specimens of aspiration are more suitable than the specimens of trephine biopsy. The frozen sections of trephine biopsy are possible and enable the application of a wide range of immunological markers. Nevertheless, the technical problems are the obstacles of using these specimens in immunological approaches.

Importance of Touch Preparation of Trephine Biopsy

Touch preparation is a procedure touching the core of bone on a slide or rolling the core gently between two slides. There are many importance of touch preparation of trephine biopsy:

• It allows cytological details to be studied since it is not possible to obtain an aspirate
• It shows more neoplastic cells than the specimens yielded by aspiration
• It may detect bone marrow infiltration which cannot detected by aspiration especially in hairy cell leukemia, multiple myeloma or lymphoma

In conclusion, although trephine biopsy is a great technique for bone marrow examination, there are many weaknesses of using this technique. The difficulty in cutting sections, poor adhesion of sections to glass slides during staining and poor morphological detail preservation are examples of the defects of this technique. Luckily, the fixed biopsy specimens that have been decalcified and paraffin-embedded without prior decalcification can help in histological examination. Next, we will discuss about the procedure to carry out trephine biopsy in the next post in Cytogenetics Cancer Research blog.

(Reference: Bone Marrow Pathology written by Barbara J. B., David M. C., Irvin A. L. and Bridget S. W.)

Trephine Biopsy | Bone Marrow Examination is a post from: Cytogenetics and Cancer Research

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