Utility of the Capsule-Based Technique for Cell Block ...

Techniques Received: May 10, 2011 Accepted: June 22, 2011 Published online: October 8, 2011

Acta Cytologica 2011;55:460–466 DOI: 10.1159/000330674

Utility of the Capsule-Based Technique for Cell Block Preparation – In Body Fluids and Liqui-PREPTM Specimens Chien-Hui Wen a Shu-Chuan Tsao a Yue-Chiu Su c Chun-Chieh Wu a Chee-Yin Chai a, b   

 

 

 

 

a

Department of Pathology, Kaohsiung Medical University Hospital and b Department of Pathology, College of Medicine, Kaohsiung Medical University, Kaohsiung, and c Department of Pathology, Foo-Yin University Hospital, Ping-Tong Hsien, Taiwan, ROC  

 

 

Key Words Cytology ⴢ Cell block technique ⴢ Body fluids

Abstract Objective: The aim of this study was to find a better technique for the cell block preparation. Study Design: We developed a novel capsule-based technique and applied it to different cell block preparation materials including plasma thromboplastin (PT) clot and agarose gel-embedding medium, in order to concentrate the cells in a limited field, then compared the result with the widely used, modified alcohol fixative preparation technique based on specimen adequacy and diagnostic accuracy. Twenty-eight nongynecologic body fluids and 41 gynecologic Liqui-PREPTM (LGM International Inc., Fort Lauderdale, Fla., USA) cytology specimens were collected for cell block preparation. We performed routine hematoxylin and eosin staining on the cell block sections, which were scored according to four specimen adequacy parameters: background, cellularity, nuclear preservation and cytoplasmic preservation. The differences between the diagnostic results of cell block slide and conventional cytology smear were also presented. Results: The capsulebased PT clot technique in nongynecologic body fluids provided more cellularity in the cell block sections and reduced

atypical diagnosis when compared to conventional smears. Conclusion: We suggest the capsule-based PT clot technique is a better technique for nongynecologic body fluids in the preparation of a satisfactory cell block. Copyright © 2011 S. Karger AG, Basel

Introduction

In routine cytological practice, the cell morphologic changes in smears are not always obvious. Sometimes judgment is difficult; therefore, in the application of cell blocks for better presentation of detailed cytological architectural features, histochemical or immunocytochemical staining has been a useful adjunct for establishing a more definitive cytopathological diagnosis [1]. Furthermore, cell block is also a source of archival material for cytological research [2]. For many years, cell block preparation techniques have been widely applied in many cytology specimens, including body fluids, fine-needle aspirations and liquid-based specimens. The techniques used for cell block preparation vary in different institutions. Several cell block preparation techniques have been introduced in order to improve the diagnostic value of histochemical

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Fig. 1. ‘Alcohol’, capsule-based PT and capsule-based ‘agar’ cell block preparation techniques. a Cell sediments in the tube base using ‘alcohol’ (left), capsule-based technique in PT material (middle) and capsule-based technique in ‘agar’ material (right). b Gross morphology of the cell clots: cell sediments of the ‘alcohol’ were soft and fragile (left), the capsule-based PT clot was soft and watery (middle) and the capsule-based ‘agar’ clot showed obvious solid cell cylinder (right).

and immunohistochemical stains [3]. The following techniques have been reported: ethanol formalin fixative [1], bacterial agar [4, 5], plasma-thrombin clot [6–9], albumin, inverted filter sedimentation [10] and simple sedimentation [11, 12]. However, these techniques still have their disadvantages; e.g., the cellular elements are usually dispersed over the optical field. In a literature review, most of the published papers revealed that cell block preparation techniques were applied in nongynecologic body fluids, aspirations and ThinPrep liquidbased cytology specimens. To the best of our knowledge, the application of the cell block technique in the LiquiPREPTM, a recently introduced liquid-based cytology, has not yet been published. In this study, we compared the quality of cell block sections that were obtained from different cell block preparation materials, including alcohol fixative (‘alcohol’), plasma thromboplastin (PT) clot and agarose gel-embedding (‘agar’) medium applied in both body fluids and Liqui-PREP specimens. We found that the cell block sections yielded more comprehensive information for a precise diagnosis, including higher cellularity and better nuclear and cytoplasmic preservation. In this article, we demonstrate not only the detailed procedures of the different types of capsulebased cell block preparation technique, but also describe their cytomorphological features in the cell block sections. We also discuss the degree of difficulty of, and the time consumed by, these cell block preparation techniques. Capsule-Based Cell Block Preparation

Materials and Methods Case Collections Twenty-eight body fluids (including 11 pleural effusion, 1 ascites, 1 bile, 1 liver, 1 pericardial effusion, 1 peritoneal washing and 12 urine specimens) and 41 gynecologic Liqui-PREP specimens were collected for cell block preparation. Each of the body fluids and gynecologic Liqui-PREP specimens was equally divided into three separated centrifuged tubes for different cell block preparation materials. Cell Block Preparation Technique A Modified ‘Alcohol’ Technique Equal amounts of 75% ‘alcohol’ were added to each specimen and mixed for 5 s. The tube was then centrifuged at 2,500 rpm for 3 min to form a cell sediment. After the first centrifuge, the supernatant fluid was decanted and equal amounts of 95% ‘alcohol’ were added to the cell sediment and mixed again and the tube was again centrifuged at 2,500 rpm for 3 min. The supernatant fluid was decanted again and the cell sediment was then carefully removed from the tube, wrapped in lens-cleaning paper and placed in a Tissue-Tek (Simport Plastic Ltd.) cassette. Capsule-Based PT Clot Technique The specimens were centrifuged at 2,500 rpm for 3 min to form the cell sediment. After the supernatant fluid was decanted, 2 drops of human plasma (Dade Behring, Marburg GmbH, Marburg, Germany) and 4 drops of thromboplastin (Dade Behring) were added to the cell sediment. The cell sediment and PT were mixed for 5 s and absorbed into a gelatin-based capsule (size ‘01’, Da-Ming Capsule Co., Ltd., Taiwan) to form a clot for 3–5 min. The cell clot was then removed from the capsule with a thin bamboo stick, wrapped in lens-cleaning paper and stored in a TissueTek cassette.

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Capsule-Based ‘Agar’ Technique All the specimens were centrifuged at 2,500 rpm for 3 min to form the cell sediment. The supernatant fluid was decanted and then equal amounts of 10% formalin were added to the cell sediment and mixed for 5 s, followed by centrifuging again at 2,500 rpm for 3 min. The supernatant fluid was decanted and 6 drops of molten 2% agarose gel were added to the cell sediment. The cell sediment and agarose gel were mixed for 5 s. In the ‘agar’ material, we mixed the cell sediment and agarose gel and absorbed it into a gelatin-based capsule (size ‘01’, Da-Ming) to form an agarcell clot cylinder for 3–5 min. The agar-cell clot cylinder was removed from the capsule and was placed in a Tissue-Tek cassette. All Tissue-Tek cassettes were immersed in 10% formalin before processing in the automatic tissue processor. The cell blocks were constructed with embedded paraffin and 3-␮m sections were cut using a standard microtome and then treated with hematoxylin and eosin stain and mounted on a glass coverslip. Scoring for the Cell Block Sections All the hematoxylin and eosin-stained cell block sections were reviewed and scored (0–3 points) according to four parameters: background, cellularity, nuclear preservation and cytoplasmic preservation. Background: 0 = area of 175% with blood/mucus/fibrin, 1 = area of 51–75% with blood/mucus/fibrin, 2 = clear with area of 25–50% with blood/mucus/fibrin, 3 = clear with area of ! 25% with blood/mucus/fibrin. Cellularity: 0 = no diagnostic cell, 1 = occasional single cell or cluster, 2 = epithelial cells easy to find, 3 = many epithelial cells in almost every field. Nuclear and cytoplasmic preservation: 0 = no preservation, 1 = poor preservation, 2 = fair preservation, 3 = good-to-excellent preservation. The final diagnoses of the cell block sections and the cytological smears were compared.

Results

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Fig. 2. a–c Capsule-based ‘agar’, ‘alcohol’ and capsule-based PT cell block sections of a pleural effusion. d–f Capsule-based ‘agar’, ‘alcohol’ and capsule-based PT cell block sections in a LiquiPREP specimen. The capsule-based ‘agar’ cell block section had a clear background but that of the ‘alcohol’ and capsule-based PT sections was pale to deep pink.

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Two hundred and five cell blocks were successfully prepared from 69 cases except for 2 cell blocks that lost their components during the cell block preparation. These ‘lost’ cell blocks were prepared with ‘alcohol’ and the components were lost during the transfer from the centrifuged tube to the lens-cleaning paper. However, the capsule-based technique, both in PT and ‘agar’ preparation materials, was successful for cell block preparation (fig. 1). Figure 2 shows the cell pattern in the sections of the 3 representative cell block preparation materials in body fluids and Liqui-PREP specimens. During the preparation of the cell block, the solid agar-cell clot cylinder could easily be removed from the capsule and transferred into a cassette, and lens-cleaning paper was not needed to prevent the loss of the cellular component. However, a thin bamboo stick was needed to remove the soft PT clot from the capsule and wrap it with lens-cleaning paper. Concerning the cellular component transfer preceding Wen /Tsao /Su /Wu /Chai  

 

 

 

 

Table 1. Scoring results of the capsule-based technique for body fluids and gynecologic Liqui-PREP specimens

Background, n

Cellularity, n

BF

LP

BF

‘Alcohol’ 0 1 2 3 N

1 (4) 26 (93) 0 1 (4) 28 (100)

1 (2) 36 (88) 1 (2) 3 (7) 41 (100)

1 (4) 3 (11) 15 (54) 9 (32) 28 (100)

CPT 0 1 2 3 N

0 28 (100) 0 0 28 (100)

0 39 (95) 0 2 (5) 41 (100)

CAGAR 0 1 2 3 N

0 2 (7) 4 (14) 22 (79) 28 (100)

0 3 (7) 3 (7) 35 (85) 41 (100)

Technique (scoring point)

Nuclear preservation, n

Cytoplasmic preservation, n

BF

LP

BF

LP

1 (2) 1 (2) 3 (7) 36 (88) 41 (100)

1 (4) 0 3 (11) 24 (86) 28 (100)

1 (2) 0 2 (5) 38 (93) 41 (100)

1 (4) 0 4 (14) 23 (82) 28 (100)

2 (5) 0 2 (5) 37 (90) 41 (100)

0 2 (7) 5 (18) 21 (75) 28 (100)

0 0 6 (15) 35 (85) 41 (100)

0 0 2 (7) 26 (93) 28 (100)

0 0 2 (5) 39 (95) 41 (100)

0 0 2 (7) 26 (93) 28 (100)

1 (2) 0 1 (2) 39 (95) 41 (100)

0 7 (25) 12 (43) 9 (32) 28 (10)

0 3 (7) 33 (80) 5 (12) 41 (100)

0 0 7 (25) 21 (75) 28 (100)

0 0 5 (12) 36 (88) 41 (100)

0 0 7 (25) 21 (75) 28 (100)

0 0 5 (12) 36 (88) 41 (100)

LP

Figures in parentheses are percentages. CPT = Capsule-based plasma thromboplastin; CAGAR = capsule-based agarose gel-embedding medium; N = total case number; n = case number; BF = body fluids; LP = Liqui-PREP.

the paraffin embedding, the solid agar-cell clot was the easiest to transfer in comparison to the fragile alcoholfixed cell sediment and the capsule-based PT. However, when the entire process was considered, the capsulebased PT technique was the simplest compared to the capsule-based ‘agar’ and ‘alcohol’ techniques. The distribution of the scoring results in body fluids and Liqui-PREP specimens is shown in table 1. The ␹2 test and Fisher’s exact test were applied to analyze the correlation between the different cell block preparation techniques and the scoring factors (data not shown). The background and cellularity factors revealed a significant difference between the 3 cell block preparation techniques (p ! 0.05). Sections prepared by the ‘alcohol’ and capsule-based PT techniques had a denser pink background and more cellularity than the capsule-based ‘agar’ technique. The cytomorphological features from all cell blocks showed good-to-excellent preservation of the nucleus and cytoplasm. The results of the comparison of the diagnosis between cell block sections and cytology smears in body fluids and gynecologic specimens are shown in table 2. In

body fluids, plasma and alcohol materials could yield a more definite diagnosis than the agar material. However, the morphology in the cell block sections of the LiquiPREP specimens did not improve the diagnostic value by using these 3 different techniques.

Capsule-Based Cell Block Preparation

Acta Cytologica 2011;55:460–466

Discussion

Cell blocking has been reported as an auxiliary tool in diagnosis by cytological smear. Especially for ambiguous lesions, cell block sections are significantly better than smears, both in providing a diagnosis or contributing additional information to establish a definite diagnosis [13]. A number of the preparation techniques of cell blocking have been reported [1, 4–6, 8–12]. In addition, the PT technique, when applied in fine-needle aspiration and serous effusion specimens, has been considered to be simpler and easier than the ‘agar’ technique [14]. To evaluate the diagnostic value of cytology smear, Nigro et al. [10] tried to use three different preparations in order to find a better technique of cell block preparation in nongyneco463

logic ThinPrep specimens and they found that the thrombin-based cell block technique was superior to inverted filter sedimentation, albumin and simple sedimentation. In this study, we attempted to develop the capsulebased technique for preparing a cell block in body fluids and Liqui-PREP specimens. The gelatin-based capsules were not expensive and they could easily be obtained from a pharmacy. We used the size ‘01’ gelatin-based pharmaceutical capsules (about 5 mm in diameter) for the study and the capsule-based technique was successfully applied in PT and ‘agar’ materials. The main advantage of the capsule-based technique was that the cell clot of the PT material and the agar-cell clot cylinder could be removed easily from the capsule and transferred to the lens-cleaning paper and cassette. In addition, the cellular component could be concentrated in a smaller optical field. Moreover, no additional tool was needed to remove the agar-cell clot cylinder from the capsule into the cassette in order to avoid further fragmentation of the agar-cell clot. Regarding four scoring factors, the background and cellularity revealed a significant difference between the 3 cell block preparation techniques (table  1). Sections of capsule-based PT and ‘alcohol’ materials showed a paleto-deep pink background, while sections of capsule-based ‘agar’ material revealed a clear background. The pink background was likely caused by the deposition of fibrinoid materials and it did not influence the cytological interpretations. The cellularity in the sections of capsulebased ‘agar’ material was less than in these sections in capsule-based PT and ‘alcohol’ materials. Most of the cells in the capsule-based PT and ‘alcohol’ cell block sections were aggregated into large clusters. On the other hand, smaller cluster cells and a diffuse cell pattern were noticed in capsule-based ‘agar’ cell block sections. We noticed that the agarose-gel volume added into centrifuged tubes should be less than the cell sediment volume. If an excessive agarose-gel volume was added, the cellular component became dispersed in the cell-clot cylinder and the cell block section showed poor cellularity. Therefore, we suggest that the addition of agarose-gel could be a crucial step in the capsule-based ‘agar’ technique. Unlike the agar-cell clot cylinder, the cell clots of PT material were flattened after processing in the automatic tissue processor and the cellular sediments of the ‘alcohol’ material were fragile. Therefore, the capsule-based PT and ‘alcohol’ cell blocks could be cut into a lesser number of cell block sections than the ‘agar’ cell block. However, all 3 cell block preparation techniques provided a good-to-excellent nuclear and cytoplasmic preservation in their sections (fig. 3, 4). Under microscopy, we found that the pat464

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Table 2. Comparison of the diagnosis between cell block slide and cytological smear in body fluids and gynecologic Liqui-PREP specimens

Technique (diagnosis)

Body fluids

Liqui-PREP

cell block, n smear, n

cell block, n smear, n

‘Alcohol’ Negative 21 (75) Atypical 1 (4) Suspicious 0 Positive 6 (21) LSIL HSIL N 28 (100)

15 (54) 5 (18) 3 (11) 5 (18) 28 (100)

CPT Negative 20 (71) Atypical 0 Suspicious 2 (7) Positive 6 (21) LSIL HSIL N 28 (100)

15 (54) 5 (18) 3 (11) 5 (18) 28 (100)

CAGAR Negative 20 (71) Atypical 1 (4) Suspicious 4 (14) Positive 3 (11) LSIL HSIL N 28 (100)

15 (54) 5 (18) 3 (10) 5 (18) 28 (100)

31 (76) 5 (12)

29 (71) 2 (5)

3 (7) 2 (5) 41 (100)

5 (12) 5 (12) 41 (100)

29 (71) 3 (7)

29 (71) 2 (5)

4 (10) 5 (12) 41 (100)

5 (12) 5 (12) 41 (100)

29 (71) 6 (15)

29 (71) 2 (5)

3 (7) 3 (7) 41 (100)

5 (12) 5 (12) 41 (100)

Fig ures in parentheses are percentages. CPT = Capsule-based plasma thromboplastin; CAGAR = capsule-based agarose gelembedding medium; N = total case number; n = case number.

tern of the cell nuclei in the capsule-based ‘agar’ cell block section was smaller and that the cells aggregated easily to form many small clusters. The morphology of the cells in the capsule-based PT and ‘alcohol’ cell block sections was similar to the cells in the conventional cytological smears. Regarding the diagnostic value of cell block sections and cytological smears, cytological smears were shifted to negative in 5 atypical cases in the capsule-based PT cell block section, and in 4 cases in the ‘alcohol’ and capsulebased ‘agar’ cell block sections. The capsule-based PT cell block section decreased 18% of atypical diagnosis when compared to cell block sections and cytological smears in body fluids (table 2). Capsule-based PT cell block section may decrease the atypical diagnosis when compared to ‘alcohol’ and capsule-based ‘agar’ sections in body fluids. But due to low cellularity, 1 suspicious and 2 positive casWen /Tsao /Su /Wu /Chai  

 

 

 

 

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Fig. 3. Cell block section of a diagnosis of adenocarcinoma in pleural effusion using 3 different preparation techniques. a Capsule-based ‘agar’ cell block. b ‘Alcohol’ cell block. c Capsule-based PT cell block. All these

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techniques revealed 3-dimensional clusters with good-to-excellent nuclear and cytoplasmic preservation.

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Fig. 4. Cell block section of a mild dysplasia (CIN 1) with koilocyte in a Liqui-PREP specimen. a Capsule-based ‘agar’ cell block. b ‘Alcohol’ cell block. c Capsule-based PT cell block.

es in the capsule-based ‘agar’ sections and 2 suspicious cases in the ‘alcohol’ sections were shifted to negative. Furthermore, 1 suspicious case in the conventional cytological smear was shifted to positive in the capsule-based PT and ‘alcohol’ sections, because these sections showed more detail of the cellular atypia and a lesser degree of cellular overlapping. In contrast, the atypical diagnosis was increased in the Liqui-PREP cell block sections. Of these 3 techniques, the diagnostic value of the capsule-based PT cell block section was similar to that of the conventional cytological smear. Due to low cellularity, 2 LSIL cases and 1 HSIL case were shifted to atypical diagnosis in the ‘alcohol’ section and 1 LSIL case was shifted to atypical diagnosis in

the capsule-based PT section, while 2 LSIL cases and 2 HSIL cases were shifted to atypical diagnosis in the capsule-based ‘agar’ section. Two HSIL cases were shifted to negative and this was due to the loss of cellular components in the ‘alcohol’ technique. Therefore, the cell block preparation techniques in Liqui-PREP specimens did not improve the diagnostic value. In conclusion, the capsule-based PT technique is a better technique in the preparation of cell block for nongynecologic body fluid specimens, yielding a higher quality cell block. Using the capsule-based PT technique in nongynecologic body fluids could obtain more cellular components in the cell block sections and reduce atypical diagnosis when compared to the conventional cytological smear.

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