Introduction
Cytology, or Cytopathology, involves examining cells from bodily tissues or fluids to reach a probable or definitive diagnosis. The benefits of this method are that it is less invasive, economical, easy to perform and can yield quicker results, to name a few.
Diagnostic cytopathology incorporates various types of techniques, like aspiration cytology (FNAC), fluid cytology, imprint cytology etc. for determination of diagnosis.
However, FNAC and other types of cytology have their own drawbacks.
Problems like poor preservation of cellular morphology, poor preservation of architecture, hemorrhagic aspirates, clumping of cells can be a hindrance in diagnosing various lesions, especially malignant ones.
This is where cell block technique comes in handy.
Cell block preparation is a well-known technique in cytological diagnosis, which acts as a bridge between cytology and histopathology. It helps to maintain architectural integrity of the tissue under study, thus aiding visualization of various patterns and morphology. It also helps in maximum preservation of cellular and nuclear morphology, thus helping in identifying nuclear features optimally.
There is no standardized method currently for preparation of cell blocks, and various methods include plasma-thrombin, Carnoy’s method, etc.
Through this study, we aim to propose the Formalin-Alcohol technique which is cost effective, consumes less time and can be performed with reagents which are easily available at the histopathology laboratory.
Materials and Methods
We conducted a study intended to compare two techniques of cell block preparation, and shed light on the utility of formalin-alcohol method of cell block preparation.
Our study was conducted over a period of two months, and included 20 samples.
The samples included in our study comprised pleural fluid, pericardial fluid, ascitic fluid and urine.
All samples were subjected to cell block preparation by two methods:
Formalin Alcohol Method and Plasma Thrombin Method.
The methodology has been described in brief below:
Formalin alcohol method
3-4 ml fluid was centrifuged; supernatant discarded and the sediment mixed with
10% formal-alcohol (90 ml absolute ethanol + 10 ml concentrated formaldehyde).
After standing for 1 hour, a clot was formed which was submitted for processing.
Plasma thrombin method
3-4 ml fluid was centrifuged; supernatant discarded and the sediment mixed with citrate plasma & thromboplastin in equal amounts.
After shaking well, the clot formed was fixed in neutral buffered formalin for 6 hours and then submitted for processing.
Following automated processing, the slides were stained with H&E stain, and microscopic examination was carried out.
Special stains were performed wherever necessary.
Results
The 20 fluids which we processed in our study, spanned over following types and categories Table 1.
Table 1
Type and category of fluid
|
Type of fluid |
Pleural fluid (n=09) |
Ascitic fluid (n=06) |
Urine (n=03) |
Pericardial fluid (n=02) |
|
Pathological process |
||||
|
Inflammatory |
03 |
04 |
00 |
02 |
|
Benign |
02 |
00 |
00 |
00 |
|
Malignant |
04 |
02 |
03 |
00 |
The following morphological parameters were studied, and graded as mentioned Table 2.
Table 2
Morphological parameters
We observed in our study that of the parameters analyzed, cell blocks prepared by formalin alcohol method showed superior quality in cellular morphology and nuclear morphology. (Figure 1)
Routine HE staining of the cell block demonstrated a clear distinction between inflammatory, benign, and malignant cells. Staining intensity was also better in cell blocks prepared by the above method. (Figure 1)
Urine cytology in particular showed a distinct improvement.
Pericardial and ascitic fluid examination also revealed similar results. (Figure 2, Figure 3)
We received pleural fluid cytology of a patient with history of carcinoma breast. Immunohistochemistry was performed on cell block tissue prepared by the formalin-alcohol method, which showed positive staining for ER & PR.(Figure 4)
Discussion
Fluid cytology, and cytology in general, is one the oldest, albeit most reliable techniques in the diagnosis of various entities.1, 2 However, failure rate in this technique is marked, due to reduced cellularity.
Cell block preparation allows for the reduction of this failure rate, by controlling the processing-related artifacts and reducing errors like pressure-induced crush artifacts, clumping due to improper spreading, etc.3 This is because the basic principle behind the cell block is transforming the fluid is transformed into an easily recognizable tissue-like pattern.4
Various methods of cell block preparation are available, like The Sodium Alginate Method, The Pregelatinized Starch Method, The Cell-Gel Method and the Histogel Tube Method etc.5, 6, 7, 8, 9
Desai et al1 in their study used the modified alcohol-formalin technique to preserve FNAC samples and to make cell blocks from various cases of head and neck pathologies. Their findings and conclusion were comparable to our study.
Nathan et al. 4, 10 in their study, suggest ed that formalin, despite being a good tissue fixative, does not preserve the cellular details in a cell block preparation. They used Nathan alcohol formalin substitute and observed a better maintenance of cellular features. Their technique, however, led to a longer processing time than our technique.
In our study, we used the modified formalin-alcohol technique to preserve and process fluid samples. This technique is a modification of the 10% alcohol-formalin technique employed by Udasimath et al. 6 However, our methodology differed from theirs in the way that we limited the use of alcohol and prolonged the fixation time with formalin.
All the samples of ascitic fluid, pericardial fluid and pleural fluid yielded results in favor if Formalin-alcohol method. 11, 12, 13, 14
In our comparison between the two methods, ie. formalin-alcohol method and plasma thrombin method, the observations we made were as follows:
Table 3
Comparative analysis of both methods
We also observed that for the purpose of immunohistochemistry, formalin alcohol method proved to be a better method, due to superior fixation of tissue.15, 16
Thus, the modified formalin-alcohol technique has numerous advantages with a few limitations.
Conclusion
Formalin-Alcohol technique of cell block preparation has proven to be a useful and resourceful method on a routine basis, due to its optimal cellular morphological appearance, cost effectiveness and ease of preparation in the laboratory.
We encourage that cell block preparation be made a routine practice whenever FNAC is indicated, and the formalin-alcohol method is a recommended method for routine use in a histopathology laboratory.
