Many physicians believe

that a pathologist looking at a slide

must see and understand everything

that is of interest to the clinician.

M. F. Glazunov, 1961

Examination of biopsy and surgical samples is a type of intravital pathological diagnosis of skin diseases and neoplasms using microscopic analysis of structural changes. Artifacts of the examined material affecting the interpretation of the microscopic pattern and the making of the pathology conclusion represent a major issue. Artifacts not only reduce the quality of slides but can also cause diagnostic errors [1]. Tissue sample processing in the pathology laboratory is a series of steps designed to remove water from biopsy and surgical sample to ensure optimal quality. The steps include fixation, dehydration, clearing, paraffin impregnation, and paraffin block embedding. Prevention of errors at the histological processing stage is an urgent practical task that any pathology laboratory has to address routinely.

Also, the quality of the sample material can be seriously impacted due to preanalytical artifacts. Inappropriate handling of biopsy and surgical samples during receipt, storage, and transportation significantly reduces the informative value of the microscopy and hinders the assessment and interpretation of abnormalities [2].

Since the accuracy of pathology examination depends on specified indications for histological examination, the choice of skin area to be biopsied, biopsy sample size, and the availability of complete clinical information, artifacts arising at the preanalytical stage can be tentatively classified as follows:

— Defects in lab slip filling;

— Defects in selection of the rash element;

— Defects during sampling;

— Fixation defects.

The dermatovenerologists and procedural nurses should be aware of these artifacts, and most importantly, these artifacts should be avoided, as they are unrecoverable during the analytical stage of biopsy material processing.

Defects in lab slip filling

The protocol of intravital pathological examination of biopsy (surgical) sample (form 014/u) is an accounting medical record approved by Order of the Russian Ministry of Health No. 179N of 24.03.2016 [3]. The protocol form consists of several modules; its front side is filled by the attending physician, where along with personal data, there is a section for clinical data. Details on the duration, nature, and localization of rash elements and skin neoplasms are necessary for the dermatopathologist and are of great importance for high-quality, effective, and timely morphological examination. In most cases in real-world practice, clinical data in the protocol form are sparse, written in illegible handwriting, or absent (Fig. 1).

Fig. 1. Defect in lab slip filling — missing data in the sections “Objective of the lifetime examination”, “Additional clinical information”, “Results of previous histological studies”.

Unfortunately, the problem of communication between dermatovenerologists and pathologists related to incomplete or inaccurate clinical data in the lab slip form for histological examination is typical not only for our country. Thus, in a survey of 1,103 members of the American Society of Dermatopathology, 42.7% of respondents rated clinical data in referrals as insufficient; 91% of dermatopathologists (out of 598) indicated that inaccurate or missing clinical data made their work very difficult; 44% of study participants reported spending more than 30 min (52% less than 30 min) per day to draw missing clinical information [4].

Defects in selection of the rash element

1. An atypical rash element was selected. For instance, in the case of a typical lesion on the face (e. g., discoidal lupus erythematosus) and atypical lesions on the trunk, most cases will be sampled from the trunk. Difficulties in the diagnosis verification may also arise from biopsies of psoriatic plaques localized in acral areas since they often show a more pronounced exudation and the absence of hypogranulosis, which hampers differentiating psoriasis from chronic eczema. In the elderly, along with the described features, histological signs of venous stasis may be present in the dermis, which may mimic the pattern of congestive dermatitis (Fig. 2). A typical cavitary element not more than 24 h of age should be biopsied for the histological diagnosis of vesicular dermatoses. Otherwise, with rapid re-epithelialization of the cavitary element, the depth of its formation cannot be reliably determined on histological examination (Fig. 3). Another example of atypical rash element selection would be a biopsy of secondary rash elements with similar morphological changes in any dermatosis (Fig. 4).

Fig. 2. An atypical rash element localized in the ankle area was selected for histological examination in a patient with psoriasis.

a — epidermis acanthosis with irregular elongation of epidermal ridges (hematoxylin and eosin (H&E) staining, ×40); b — coagulated plasma inside the stratum corneum (green arrow), differentiated granular layer (red arrow), the “balls” of vessels with a thickened wall in the dermis simulates the pattern of chronic eczema combined with signs of venous stasis (yellow arrow) (H&E staining, ×100).

Fig. 3. A patient with vesicular dermatosis had a biopsy of an “old” vesicular element.

a — a cavity (green arrow) in the center of the dissected fragment (H&E staining, ×40); b — the blister cap represented by the necrotic epidermis (red arrow), re-epithelization in the marginal zones (yellow arrow). The level of vesicle formation and the presence or absence of acantholysis could not be determined from this biopsy specimen (H&E staining, ×100).

Fig. 4. Secondary element of the rash.

a — rash element with an excoriated area (H&E staining, ×40); b — the integrity of the epidermis is compromised, which precludes assessment of changes within the epidermal layer (green arrow) (H&E staining, ×100).

2. An immature rash element was selected. In some cases, according to the pathological process course, it takes some time to form a developed pathomorphological pattern. Therefore, typical findings are not always fully formed in new rash elements. Thus, if morphological verification of granulomatous dermatoses is required, the "oldest" rash element should be selected for biopsy. Otherwise, pathological findings may be nonspecific (Fig. 5).

Fig. 5. Lack of a clear morphological pattern.

a — an emerging granuloma with annular granuloma (H&E staining, ×40); b — mild infiltration between collagen bundles in the reticular layer of the dermis (green arrow) (H&E staining, ×100).

Biopsied rash element demonstrates treatment-related pathomorphosis. Treatment-related pathomorphosis develops quickly, especially when treated with topical corticosteroids. Such artifacts are due to the availability of topical corticosteroids for self-administration by patients and the histological study performed after the treatment. In such a situation, histological examination becomes meaningless, since specific morphological changes resolve very quickly with therapy, and the morphological pattern acquires the features of nonspecific dermatitis (Fig. 6). To avoid this, biopsy material should be sampled before treatment or therapy should be interrupted for at least two weeks, or sampling should be performed at a subsequent exacerbation.

Fig. 6. Morphological pattern with signs of nonspecific dermatitis.

a — therapeutic pathomorphism due to topical glucocorticosteroids (H&E staining, ×40); b — mild acanthosis of epidermis (green arrow), scanty inflammatory infiltration (red arrow). No specific features identified (H&E staining, ×100).

Defects during sampling

1. Biopsy specimen was taken at the border of intact skin. For some reason, most dermatovenerologists have a strong belief that material should be taken "at the border of intact skin" or "with the capture of intact skin." This belief probably originated from oncology, where it is crucial to excise the tumor within intact tissue with histological evaluation of the resection margins, as it is relevant for pathohistological staging. The histological diagnosis of chronic dermatoses requires a different approach. Since the sample size is usually small, it is preferable to have as much abnormal tissue as possible in the sample. However, when sampling is performed at the border of intact skin, abnormal tissue is usually absent in most of the sample (Fig. 7). Moreover, when local anesthesia is used, particularly with vasoconstrictor drugs, it is not always possible to determine the boundary between affected and intact skin at the time of the biopsy procedure. Thus, the major part of the biopsy sample is usually comprised of intact skin with no evidence of abnormalities. Therefore, to avoid such errors, the sample should be taken from the center of the lesion.

Fig. 7. Biopsy specimen taken at the border of intact skin.

a — most of the biopsy specimen is intact skin, no abnormalities observed (green arrow) (H&E staining, ×40); b — mild inflammation in the marginal zone of the biopsy specimen (red arrow) (H&E staining, ×100).

2. The biopsy was taken superficially. Suspected diseases should be considered during sampling. For instance, for lichenoid dermatoses, it is sufficient that the biopsy specimen contains the epidermis and a small section of the dermis. In cases where abnormalities in the deep dermis or subcutaneous adipose tissue need to be verified, a superficial biopsy will not be sufficient, and the sample will also be non-diagnostic (Fig. 8).

Fig. 8. Superficial biopsy.

a — superficial biopsy taken, deformed skin fragment (H&E staining, ×40); b — skin fragment represented by epidermis and thin dermis layer without underlying tissues (H&E staining, ×100).

3. Sampling was performed with an electrocoagulator (cauterization artifacts). Tissue should be dissected during the biopsy procedure using sharp cutting instruments (scalpel, circular knife). However, it is not uncommon in clinical practice for dermatovenerologists to collect material using different equipment, electrocoagulator, high-frequency radiosurgical device, etc. This is probably due to the convenience of sampling and minimizing bleeding. However, the sample obtained in this way becomes completely unsuitable for histological examination due to the marked damage resulting from electrical trauma (Fig. 9). This applies not only to dermatoses but also to skin neoplasms because artifacts resulting from electrical trauma prevent assessment of tumor cell maturation degree and neoplasms histogenesis, which, in turn, makes it impossible to make a life prognosis for the patient.

Fig. 9. Specimen sampling was performed with an electrocoagulator.

a — skin fragment with marked cauterization artifacts (H&E staining, ×40); b — cell deformation resulting from electrical injury (green arrow) precludes accurate assessment of the pathological process (H&E staining, ×100).

4. Mechanical damage artifacts occur most often when a biopsy sample is crushed by forceps, which hampers microscopic diagnosis because of the almost complete loss of microscopic structure (Fig. 10).

Fig. 10. Artifacts of mechanical injury.

a — biopsy specimen squeezed by forceps during material sampling (H&E staining, ×40); b — cell deformation due to tissue crush (green arrow) precludes accurate assessment of the inflammatory infiltrate cellular composition (H&E staining, ×100).

5. Prefixation dehydration artifacts occur during prolonged storage of the biopsy sample in air (out of fixation solution). Due to tissue drying and coagulation necrosis, morphological verification of the disease becomes impossible (Fig. 11).

Fig. 11. Prefixation dehydration artifacts.

a — biopsy specimen placed in the fixative fluid after air drying (H&E staining, ×40); b — deformation, rupture, and coagulation necrosis of collagen bundles (green arrow), the accurate assessment of the inflammatory infiltrate cellular composition is impossible (red arrow) (H&E staining, ×100).

Defects during fixation

Immediately after the biopsy procedure, the excised fragment should be placed in a container with fixation solution. The purpose of fixation is to preserve morphological features in the examined samples. The quality of fixation can be affected by the sample size, the duration of fixation, the fixation temperature, and the fixation solution concentration. Traditionally, a 10% solution of neutral buffered formalin is used as a fixation solution. It should be considered that for proper fixation, the volume of the fixation solution should be at least ten times the volume of the skin sample. Since the penetration rate of the fixation solution into the tissue at room temperature occurs at a rate of approximately 1 mm/h, fixation time should be extended if the sample size is large, or the material should be subjected to additional fixation in the pathology laboratory. It should be noted that the penetration rate of the fixation solution into the tissue slows significantly when the ambient temperature decreases, for example, when placing containers with biopsy and surgical material in a refrigerator, which can result in so-called “cold autolysis.” Biopsy and surgical sample should be stored at room temperature until transferred to the pathology laboratory.

Another factor that can adversely affect the sample fixation is an increase or decrease in the concentration of fixation solution. If the concentration of the solution is increased, coagulation necrosis of the tissue develops; if the concentration is decreased, the tissue will undergo autolysis (decomposition) (Fig. 12).

Fig. 12. Fixation defects.

a — the surgical specimen was placed in a container of improper size; fixation was performed in a refrigerator at 4°C in an insufficient volume of fixing solution; b — autolysis of surgical specimen.

Another type of artifact arising during fixation is fixation deformation of the biopsy sample, which occurs because the skin, being soft tissue, deforms when placed in the fixation solution. Subsequently, this prevents the correct sample position in the paraffin block and adversely affects the microscopic examination process (Fig. 13). Such deformation does not occur during sampling with a circular knife (puncher). It is possible to avoid fixation deformation during scalpel excision by stretching the excised skin sample on a piece of cardboard or heavy paper before placing it in the fixation solution (Fig. 14).

Fig. 13. Fixation deformation of a skin biopsy specimen.

Fig. 14. Proper specimen collection.

a — skin biopsy specimen dissection with a scalpel; b — stretching of the dissected biopsy specimen on a piece of cardboard to prevent fixation deformation.

Therefore, healthcare professionals who perform sampling and storage of skin biopsy samples should clearly understand that many preanalytical factors, including inadequate choice of rash element and sampling technique, adverse ambient factors impacts on tissue samples, inadequate fixation, etc., can significantly impair the quality of histological examination and make it impossible to make a morphological verification of the diagnosis. Thus, the indications for biopsy and optimal sample type should be determined before the biopsy is performed. The key to successful histological examination at the preanalytical stage is a careful selection of the rash element for biopsy, proper technique of skin biopsy, and adequate fixation of the sampled material.

The authors declare no conflict of interest.