BIOPSY TECHNIQUE | Wounds Africa

Taking a biopsy of a chronic wound

Health personnel treating chronic wounds must be aware that there are clearly defined situations when a wound biopsy is indicated. This may often be to rule out malignant causes of an ulcer or to see if there are histological changes that might indicate other causes like vasculitis or pyoderma gangrenosum.

Figure 1 It is easy to learn the correct technique to biopsy a chronic wound. The biopsy sample is usually preserved in formalin 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 mechanical injury, 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 essential 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 six weeks of what seems like adequate treatment.

All chronic wounds which are older than six 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 everybody's time and resources. We have often walked into a trap ourselves- treating what we thought was a regular venous ulcer, which turned out to be a squamous cell carcinoma, for example. Remember that chronic ulcers can develop malign cells over time - 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 you should definitely get the patient referred to a higher level of care in severe cases.

Figure 2 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 biopsy should be made by a medical doctor. The procedure can be performed by a medical officer or nurse, provided that they have the necessary training and that a supervising medical doctor can 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 before attempting this. It is a straightforward procedure, and complications are rare, but bleeding or nerve damage are possible complications.

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

Since we mostly use relatively 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 ephedrine/adrenaline to reduce the amount of bleeding.

In patients using only acetylsalicylic acid, we can always take the biopsy. With concern to 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 continuous minutes (or more). Be prepared that the bleeding can last longer than expected, and you may have to place a suture in the biopsy hole to stop the bleeding.

Anticoagulants like Clopidogrel, which directly inactivate platelets, are challenging when taking biopsies. We have, on many occasions, had problems with bleeding from the biopsy sites in patients using this type of anticoagulation. Patients using this type of medication often have a more serious condition that warrants using this type of anticoagulant, and often you cannot just pause it. Sometimes we have to consult a cardiologist beforehand to hear if we can safely pause the medication before taking a biopsy.

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 made that mistake. Some patients using warfarin are poorly regulated, and we require an INR test not older than 24 hours before doing a biopsy. If the patient has an INR value in the usual therapeutic range between 2-3, we usually will perform the biopsies provided that we only are using 3mm punches.

On rare occasions, some patients even use two strong types of anticoagulants together - for example, Clopidogrel and a NOAC anticoagulant. Do not take biopsies in this case- you can likely get a bleeding that is hard to control. Again, take our word for it because we have been there. You will have to refer the patient to the specialist who prescribed these anticoagulants and inform him that you would like to do a biopsy. The specialist can then decide whether it is safe to pause at least one of the anticoagulants or to switch to another type.

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 placing pressure on the site for a longer period if the bleeding won't stop.

We routinely let patients who are on anticoagulation and got a biopsy done - sit in the waiting area for about 30 minutes afterward 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 antiseptics to clean the skin around the wound edges, swabs, local anesthesia ( lidocaine 10mg/ml), syringe and needle ( a thin needle - for example 25G is enough, biopsy punch, fine forceps, fine scissors ( optional), scalpel blade nr 15. (optional) , container with 10% Formaline (4% formaldehyde):

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 typically want to include some of the skin in 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 a local anesthetic and accurately hit the identical spot again when using the biopsy punch.

In most cases, you need to numb the areas where you plan to take a biopsy. In patients with 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 available also in Africa. If a patient uses anticoagulation medication, it is advisable to use local anesthesia with adrenaline/epinephrine to minimalize bleeding. However, the use of the vessel constricting substances may affect the way the pathologist interprets the findings, so whenever possible, avoid using adrenaline/epinephrine. Use the thinnest needle 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, and usually, less than 0,5 ml at each site is sufficient. It is far easier to inject the fluid when you have a small and thin syringe when using a thin needle. 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 favorable for most cases. The larger the biopsy- i.e., the larger the biopsy punch diameter, the easier it is for the pathologist to diagnose the ulcer precisely. However, a large diameter biopsy will also leave behind a large hole, giving you bleeding issues and may heal slowly. So we tend to compromise- using a smaller diameter punch- often 3mm or 4mm and instead 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 helpful 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 concerning 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 poisonous. 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 types of biopsies, 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 patient's name/ social security number. If you have a colleague close by, it is a good routine to double-check the patient's name and what you have written on the container. Mixing up someone's 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 anesthesia at several sites. Inform the patient beforehand that it is expected that some bleeding can occur and that you will have to apply pressure to the biopsy sites for some minutes afterward. If you fail to give this information beforehand, the patient may think you made a mistake when it started 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 plan how to inform the patient- should they come back to you when the test results are back, or will you phone them? As a general rule - if the test results show a malign cause, it is usually better to inform the patient face to face since they may get very worried. If the patient lives far away, 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. In fact, 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 can more easily see changes in the tiny blood vessels surrounding the ulcer. In some cases, however, you may get the best sample from the middle of the ulcer. We, therefore, routinely take multiple samples from the edge of the ulcer and always include one biopsy from the center of the ulcer.

Figure 4 This chronic wound looks relatively harmless, but it has not decreased over eight weeks 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 center of the wound.

Before we take the biopsies, we have to 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 no use taking a biopsy in an area with much necrotic tissue. Another consideration to make when taking biopsies are anatomical regions. Is there a larger artery or nerve close to where you plan your biopsy? You may have to consult with an anatomical atlas before doing the procedure. For example, when taking biopsies from a venous ulcer, 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 using a biopsy punch, place this at a 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 important to use the whole length of the cutting 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 index finger while rotating back and forth as you advance the punch deeper. copyright Hermann, Rauscher, 1992

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 sample is still in the hole created by the punch, and you can grasp it carefully with fine-toothed forceps and gently pull it out. Try not to squash the sample, as this will give the pathologist a headache. We often use a technique 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 an 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. copyright Lippert, 1996

Important! - do NOT be tempted to take the tissue sample straight from the forceps to your container with formaldehyde before taking all samples! You will most likely contaminate your forceps with formaldehyde and carry this back to the wound as you pick out the following 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. The tissue bits often 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 wound 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. The defects left behind by the biopsies tend to heal well within 1-2 weeks; oddly enough, even in 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 will 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 whom they use for analyzing biopsies.

Are you not happy with the results?

Quite often, 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 background for the ulcer to be a malignancy, 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 complex cases, it may be wise to take new samples and send them 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 challenging the field of pathology can be.

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

Our point here is: do not always trust a pathology report blindly. If an ulcer is not responding and 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 that summarizes the safety measures you have to take when using this chemical substance.

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