Taking a biopsy of a chronic wound 

Health personel treating chronic wounds must be aware that there are clearly defined situations when a biopsy of the wound is indicated.  Often this may be to rule out malignant causes of an ulcer or to see if there are histological changes that might be indictive of other causes like vascultitis or pyoderma gangrenosum. 

Figure 1 It is easy to learn the correct technique to do a biopsy of a chronic wound. The biopsy sample is usually preserved in formaline on the way to the pathological laboratory. 

It is important to provide the pathologist with as much information about the ulcer as possible. Do not write " we would like a routine histology performed" on the requisition.  Instead write something about what you might expect the cause of the wound to be - for example: " we cannot rule out vasculitis". This will give the pathologist some clues for what to look after.  The pathologist will also be interested in the history of the ulcer- how long has it existed, how did it start- was it a trauma or does it look like a venous ulcer? Has the ulcer increased rapidly in size? Has it been resistant to all sorts of different treatments?  Information on whether the wound is painful or not may also provide the pathologist with important clues. .

When should you perform a biopsy?

  • All chronic wounds where malignancy cannot be ruled out

  • All chronic wounds which are not responding as expected

  • All chronic wounds which are not showing signs of improvement within 6 weeks of what seems like adequate treatment.

  • All chronic wounds which are older than 6 months 

If you adhere to these recommendations you will most likely be on the safe side.  Not following these clear and easy recommendations might mean that you are wasting everybodies time and resources.  We have many times walked into a trap ourselves- treating what we thought was a regular venous ulcer which infact turned out to be a squamous cell carcinoma for example. Remember also that chronic ulcers can over time develop malign cells - this phenomenon is named Marjolin`s ulcer. 

In some parts of Africa it may not be realistic to get a histological examination performed. In these cases you may have to skip routine biopsies of ulcers but in serious cases you should definitely get the patient referred to a higher level of care. 

Figure 3  Sometimes it is easy to see that something is unusual about an ulcer, at other times the suspicious signs may be harder to spot. All the chronic wounds in this image are examples where we would do a biopsy.

Can a medical officer or a nurse perform a biopsy? 

In western countries the indication for performing a biospy should be made by a medical doctor.  The procedure can be performed by a medical officer or nurse provided that he/she has the necessary training and that a supervising medical doctor is able to aid in the procedure if necessary.  These regulations apply to ideal conditions where these resources are available.  Obviously in some parts of Africa one may have to bypass these recommendations. However, if you have never performed a biopsy before you should seek training prior to attempting this.  It is an easy procedure and complications are quite rare but bleeding or nerve damage are possible complications. 

 

Can the biopsy be done if the patient is using anticoagulation? 

 

Since we mostly use quite small biopsy punches ( usually 3mm diameter) bleeding is not usually a big problem even when patients use anticoagulation. In most cases the patient will not have to take a break from the anticoagulants - but there are exceptions.  When a patient uses anticoagulation we usually recommend using local anesthesia containing epehdrine/adrenaline as this will reduce the amount of bleeding. 

 

In patients using acetylsalicyclic acid , clopidogrel or one of the newer  NOAC ( novel oral anticoagulants) like Xarelto, Eliquis and Pradaxa we usually perform a biopsy without pausing these medications. In most cases the bleeding after the biopsies is acceptable and can be controlled by compressing the biopsy area with a cotton pad for three continous minutes. 

 

However, if the patient is using warfarin (Marevan) you should check the INR levels beforehand! You certainly do not want to take a biopsy of a patient with an INR of 5!  That will most likely give you a bleeding problem.  Take our word for it - we have done that mistake. Some patients who are using warfarin are poorly regulated and we require a INR test not older than 24 hours prior to doing a biopsy.  If the patient has a INR value in the usual  therapeutic range between 2-3 we usually will perform the biopsies provided that we only are using 3mm punches. 

Again, put some pressure with a gauze or swab on the biopsy area for a minimum of three minutes. Do not be tempted to take a look before at least three minutes have passed otherwise you will have to start all over again. If the bleeding has not stopped put pressure on the site for another three minutes. It will stop eventually but you may have to repeat putting pressure on the site for a longer period if the bleeding wont stop. 

We routinely let patients who are on anticoagulation and got a biospy done - sit in the waiting area for about 30 minutes afterwards to make sure that the site does not suddenly start to bleed again. Also, give the patient instructions to put pressure on the site for about 20 minutes if the biopsy sites should start bleeding once they have come home. 

Which tools do you need to perform a biopsy?

Figure 3  To perform a biopsy correctly you need the following tools: Non-sterile gloves, a marking pen ( optional), saline to wash the wound itself, chlorhexidine 0,5 or other antiseptic to clean the skin around the wound edges, swabs, local anestheisa ( lidocaine 10mg/ml), syringe and needle ( a thin needle - for example 25G is enough, biopsy puch, fine forceps, fine scissors ( optional), scalpel blade nr 15. (optional) , container with 10% Formaline (4% formaldehyde): 

Taking a biopsy from chronic wounds is not a sterile procedure - it is sufficient with non-sterile gloves.  You do have to use gloves and you should use an aseptic technique because you do not want to introduce new  bacteria into the wound. The biopsy punch and other sharp surgical equipment should be sterile. Clean the wound with saline or super-oxidized water. This is done because you do not want to force a large number of bacteria into deeper structures when you press the biopsy punch into the wound. The skin on the wound edges is usually prepped with chlorhexidine 0,5% or other mild antiseptic solutions. This is because we usually want to include some of the skin into the biopsy.

It is wise to mark the spots where you want to take the biopsies with a special medical marker or a regular marker. This makes it easier for you to mark the exact spot with local anesthetic and to accurately hit the identical spot again when using the biopsy punch. 

 

In most cases you need to numb the areas where you are planning to take a biopsy. In patients who have peripheral neuropathy ( for example patients with diabetes or leprosy) this may not be necessary if the skin around the lesion is without sensory function.  Lidocaine 10mg/ml is usually readily availabel also in Africa. If a patient uses anticoagulation medication it mmay be advisable to use localanethesia with adrenaline/epinephrine to minimalize bleeding.  The use of the vessel constricting substances may however affect the way the pathologist interprets the findings so whenever possible avoid using adrenaline/epinephrin. Use the thinnes needle you have to inject the local anesthetic as this is less painful for the patient.  You often only need a tiny amount of local anesthetic at each biopsy site, usually less than 0,5 ml at each site is sufficient. When using a thin needle it is far easier to inject the fluid when you have a smalland thin syringe.  For example - a 25G needle and 20ml syringe will be a poor combination - you will struggle to press the fluid through the needle.  Using a 1ml syringe with a 25G needle is ideal and you will notice immediately that it requires a lot less strength to inject the anesthetic.  Inject the anesthetic slowly- this is less painful to the patient. 

if you do not have access to a biopsy punch you can excise some tissue with a scalpel and forceps. However- a biopsy punch will give a more precise biopsy  and is favourable for most cases.  the larger the biopsy- i.e. the larger the biopsy punch diameter the easier it is for the pathologist to make a precise diagnosis of the ulcer.  However a large diameter biopsy will also leave behind a large hole which can give you bleeding issues and may heal slowly.  So we tend to compromise- using a smaller diameter punch- often 3mm or 4mm and rather taking several samples. Note: a 3mm biopsy punch gives the pathologist twice as much tissue as a 2mm biopsy punch.  We are not very good at mathematics but we understand that this has to do with the area of a circle which has the formula  pi times the radius squared (A = π r²). You can do the mathematics here if you do not believe us.

As we retract the biopsy punch the cylindrical tissue which has been excised often hangs at the base. here very fine, toothed forceps are useful for taking the cylindrical tissue out from the biopsy hole. Another tip is to use the 25G needle to lift the biopsy out of the defect and then cut it off at the base with fine scissors. 

The biopsy is placed in a container with 10% formalin (4% formaldehyde). At some workplaces  pre-filled containers with formalin/formaldehyde may be available.  This is the easiest and safest with respect to you and your colleagues.  If you do not have access to pre-filled formalin/formaldehyde containers you will have to do this yourself.  Be aware that formaldehyde is very posionous. Read the safety instructions for the product beforehand. use good ventilation while working with the product.  You do not need to fill a whole glass with formalin/formaldehyde- for these type of biopsis the container need not be filled to more than 1/3. make sure to tighten the screw cork well.  label the container clearly with the date and patients name/ social security number. If you have a colleague closeby it is a good routine to double check the patients name and what you have written on the container.  Mixing up someones biopsies is a mistake that can have drastic consequences. 

What is the correct method to perform a biopsy ?

Explain to the patient why it is necessary to perform a biopsy and how this is done.  Explain that you have to take multiple samples and inject local anestheisa at several sites. Inform the patient also beforehand that it is normal that some bleeding can occur and that you will have to apply pressure to the biopsy sites for some minutes afterwards.  If you fail to give this information beforehand then the patient may think that you did a mistake when it starts to bleed. Inform the patient that the samples have to be sent to a pathologist and that it usually takes a week ( sometimes longer) before the samples have been examined and a diagnosis is made. You also need to make a plan on how to inform the patient- should they come back to you when the test results are back or are you going to phone them.  As a general rule - if the test results show a malign casue then it is usually better to inform the patient face to face since they may get very worried. If the patient lives far away then you will probably have to phone them to convey the test results.  

To take a representative biopsy when using the smaller biopsy punches you should aim at getting at least three samples from a chronic wound.  You would think that you will get the best samples from the middle of the ulcer, right?  However, pathologists advise us to take the majority of the biopsies from the edge of the ulcer - the best technique is where you also include a tiny bit of the assumed healthy skin edge into the biopsy.  Infact, for some conditions like vasculitis the pathologist prefers it if we have included a lot of the skin edge in the biopsy and only a small part of the wound edge. In this way the pathologist more easilty can see changes in the tiny blood vessels surrounding the ulcer. In soem cases however, you may get the best samle from the middle of the ulcer.  We therefore routinely take multiple samples from the edeg of the ulcer and always invlude one biopsy from the centre of the ulcer.  

Figure 4  This chronic wound looks quite harmless but it has not decreased over a 8 week period despite adequate dressings. We therefore find it indicated to perform a biopsy.  The image on the right shows how we have planned our biopsies - three biopsies from around the edge of the wound and one biopsy from the centre of the wound. 

before we take the biopsies we have to make a plan where around the wound edges we want to take the biopsies from. Often this is determined by how the tissue looks- some areas may seem more suspicious than others. It is of no use taking a biopsy in an area with a lot of necrotic tissue. Another consideration to make when taking biopsies are anatomical regions. Is there a larger artery or nerve close to where you are planning on doing your biopsy? You may have to consult with an anatomical atlas before doing the procedure. When taking biopsies from a venous ulcer for example you will want to avoid the areas where you can clearly see varicose veins or other enlarged veins. 

It is advisable to mark the areas to be biopsied with a marker beforehand. Inject 0,5ml lidocaine 10mg/ml at each site. wait for at least 5 minutes for the local anesthesia to take effect. 

When you are using a biopsy punch place this at an right angle to the skin at the marked area. Hold the punch between the thumb and your index finger and with only slight pressure rotate the punch back and forth while gently advancing it deeper.  The punches are designed so that you cannot go too deep ( unless you use excessive force). It is imortant to use the hole length of the cuting part of the punch- i.e it has to be advanced deep enough - into the subcutaneous fat layer.  

Figure 5  A biopsy punch is designed so that it cannot be advanced too deep unless you are using excessive force. Hold the punch between your thumb and your index finger while rotating back and forth as you advance the punch deeper. 

Pull out the punch using the same rotational movements. A tip is to stretch the skin apart with your other hand as you are retracting the punch ( image A in figure 6) - in theory this may increase your chance of having the sample along in the punch on the way out - if you are lucky.  Most often however, the tissue samople is still in the hole cerated by the punch and you can grasp it carefully with fine toothed forceps and gently pull it out. Try not too squash the sample as this will give the pathologist a headache.  A technique we often use is to pierce the sample with the needle we used to inject the anesthetic - lift it out of the wound with the needle and cut it off at the base with fine curved scissors or a nr . 15 scalpel blade (image B in figure 6). 

Figure 6 A) You can try stretching the skin with your free hand while retracting the biopsy punch- this may increase the chance of the tissue sample coming out with the punch. B) A useful technique to retrieve a tissue sample is to pierce it with a needle- stretch this upwards and cut it off at the base with fine curved scissors.  

Important! - do NOT be tempted to take the tissue sample straight fromt the forceps to your container with formaldehyde before you have taken all samples!  You will most likely contaminate your forceps with formaldehyde and carry this back to the wound as you pick out the next tissue sample with the same forceps.  Retrieve all your biopsy samples from the wound first- place them on a clean surface and when you are done you can use your forceps to deposit them in the container with formaldehyde.  You can usually send all tissue samples in the same container.  

Figure 7  Do NOT contaminate the forceps with formaldehyde until you have taken all the  tissue samples from the wound. very often the tissue bits hang on to the toothed forceps and you have to shake the forceps a bit on the edge of the container making it very likely that  you can contaminate the forceps with the chemical.  For this reason- take all your tissue samples from the woudn first and only then transfer them to the container with formaldehyde. 

Some close the biopsy site routinely with a suture.  If you are using a 3mm or 4mm biopsy punch this is not necessary.  We have been doing biopsies with these diameter punches for over twenty years now and we have never closed them with a suture- ever.  The defects left behind by the biopsies tend to heal well within 1-2 weeks, oddly enough even i recalcitrant ulcers. 

As we mentioned earlier - it is quite normal to encounter some bleeding from the biopsy sites. Apply some pressure on a gauze/swab for at least three minutes. Do not be tempted to take a look before a whole three minutes have passed. it is amazing how long three minutes seem when you are doing this.  If it is still bleeding after three minutes repeat this step for another three minutes - by now the bleeding will have stopped in most cases. 

How long will it take before you get the test results?

This wil vary a lot on which pathology laboratory you are using and whether you can deliver it directly or if you have to send the sample by mail. It will also depend on what you wrote on the requisition - here you can for example specify that you would like the sample to be tested sooner because you suspect malignancy.  Even in Norway (where we have our workplace) in a public hospital routine biopsy samples often take 14 days before the results are ready.  If you live in a larger city in Africa  where you have access to a private pathology laboratory the results may be ready within a couple of days!  By the way - we have met some excellent pathologists in Africa in remote clinics in off the grid areas!  So it may be wise to check around at local clinics/ dispensaries who they use for analyzing biopsies. 

 

Are you not happy with the results?

Quite the biopsy results from a chronic ulcer show " unspecific chronic inflammation".  This can be a good sign - because unspecific chronic inflammation is what we would expect from a chronic ulcer.  However, if you still suspect the backround for the ulcer to be a malignancy then you may have to ask the pathologist to recheck the sample or take new biopsies! 

There are great pathologists and there are mediocre pathologists, just like in any other profession. In difficult cases it may be wise to take new samples and send it to another pathology lab if that is available in your area. Even the pathology lab at our  university hospital - where they have many pathologists - sometimes send samples to another clinic for a second opinion - underlining just how difficult the field of pathology can be. 

Conditions like vasculitis and especially pyoderma gangrenosum may only present with discrete histological characteristics and may be dificult to diagnose.  Some pathologist say that pyoderma gangrenosum is not a histological diagnoses becuase you have to see the whole clinical picture to make the diagnosis. Other pathologists say that they often can make this diagnose if there is sufficient tissue sample. 

Our point here is: do not always trust a pathology report blindly.  If an ulcer is not responding - rapidly increasing in size but the histology report only shows  " unspecific chronic inflammation" you should have a low threshold for taking new biopsies just to be on the safe side.

 

Hazards and precautions when using formaldehyde

Figur 8  4% Formaldehyde is the same as Formalin 10%. The substance is toxic to tissue and is cancerogenic! You should only handle this chemical after appropriate training.

Formalin (Formaldehyde) is a toxic and cancerogenic substance.  It has to be handled with extreme care and all employees handling the chemical must have appropriate training. Never smell the container - you can damage your sense of smell permanently!  Below is a link to a pdf file which summarizes the safety measures you have to take when using this chemical substance. 

wounds africa formaldehyde.JPG

Figure 9 Click on the image above to go to a link to a pdf file concerning hazards and precautions when using formaldehyde from the University of California. 

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