Evaluation of Use of RPMI Medium to Preserve Cell Morphology for Pleural/Peritoneal Fluid Cytology.

Introduction: Cytologic evaluation is the best way to detect the presence of malignancy in body cavity fluids. Roswell Park Memorial Institute (RPMI) medium or RPMI 1640 is used in cell culture, tissue culture, and also to improve the cellularity and morphology of CSF cytology specimens.
Objectives: To determine whether RPMI medium can be used to preserve cell morphology in pleural/peritoneal effusion samples. Method and Material: The study was conducted on 30 pleural/peritoneal fluid samples received routinely during 2 months for diagnostic purposes in our department. The samples were divided into four parts. One-fourth of the sample was directly refrigerated and the other fourth was at room temperature. In the other two parts, an equal volume of RPMI media was added, and one was kept at room temperature and the other refrigerated. These cytospin-prepared Giemsa-stained smears were examined for cell morphology, cellularity, and occurrence of bacterial colonies at 24 h, 48 h, 72 h, and 1 week, respectively. Result: Refrigerated RPMI medium is the best preservative for pleural/peritoneal samples; however, samples with RPMI at room temperature were equivalent/even worse than the simple refrigerated sample. Copyright:

INTRODUCTION

Cytological evaluation of the body fluids is an indispensable technique to detect malignant involvement of body cavities. The accuracy of diagnosis depends on the preservation of cell morphology which deteriorates rapidly with any delay. However, delay in transportation is inevitable due to limited working hours, thus preservation methods need to be adopted.[1]

Though current preservation methods involve conventional refrigeration at 4°, but during transportation, refrigeration is difficult. In such circumstances, the best way to preserve the specimen is not clear. Most texts have no specific recommendations.[23]

Roswell Park Memorial Institute (RPMI) medium or RPMI 1640 is a form of medium used in cell culture and tissue culture. It has been used for growing a variety of mammalian cell lines and growth of human lymphocytes.[45] The RPMI medium has been previously used to improve the cellularity and morphology of CSF cytology specimens.[6] But no previous study exists on its use as a preservative for pleural or/and peritoneal fluids.

We hereby present a study wherein RPMI was utilized in an attempt to preserve body fluids.

MATERIAL AND METHODS

The study was conducted on 19 peritoneal and 11 pleural fluid samples received routinely during 2 months for diagnostic purposes in our department. Approval from the ethics committee had been obtained dated 27/5/2019, F.17/IEC/MAMC/19/No.122.

The samples preferably more than 20 mL were divided into four parts. One-fourth of the sample was directly refrigerated and the other fourth was kept at room temperature. In the other two parts, an equal volume of RPMI media was added, and one was kept at room temperature and the other refrigerated. All the samples were run at 24 h, 48 h, 72 h, and 1 week, respectively. The slides were examined by two independent pathologists, and the data were recorded separately (with 100% concordance). These slides were examined for cell morphology, cellularity, and occurrence of bacterial colonies in cytospin-prepared Giemsa-stained smears.

Morphological cell changes (presence of cytoplasmatic blebs and vacuoles, nuclear pyknosis, and chromatin alterations) were investigated for all slides (10 fields/slide at 100 ×– magnification). The changes were semi-quantified according to the following score.[7]

(0)no morphological changes;

(1+) from 1 to 10 morphological changes in a median of 10 analyzed microscopic fields;

(2+) from 11 to 30 changes; and

(3+) more than 31 morphological changes.

Cellularity was documented as the number of cells was counted, and the percentage of reduction of cellularity from the original sample was noted.

1- <25% reduction

2- 25%–50% reduction in cellularity

3- 50%–75% reduction

4- >75% reduction.

Bacterial colonies count

None 0few 1+dense 2+

The results were analyzed using Fisher's exact test on the four samples at different time intervals (SPSS software), and P value was obtained.

No additional sampling for the study was performed on patients. Diagnosis of cases was not compromised, and routine processing of the sample was carried out side by side.

RESULTS

From a total of 30 samples that were received, 19 were peritoneal and 11 pleural fluids. The male to female ratio was 14:16. All the samples were divided into four sets and processed and analyzed at 24 h, 48 h, 72 h, and 1 week, respectively. Morphological analysis of the cells revealed nuclear changes and cytoplasmatic changes [Figure 1a] in the majority of cell types ofall the samples analyzed at 24 h duration highest in samples at room temperature followed by RPMI at room temperature, and least in refrigerated RPMI samples. As shown in Table 1, RPMI at room temperature showed much more morphological changes compared to other groups after 48 h, 72 h, and 1-week time duration. Morphological changes were more or less similar in samples at room temperature with or without RPMI. However, the samples that were refrigerated with RPMI had a slightly better outcome as the less morphological score at the respective time duration. While correlating the four groups through Fisher exact test, we found a significant difference between these groups. It is important to highlight that in effusions fluids, the identification of malignant cells was reliable up to 72 h after collection in samples stored under refrigeration with or without RPMI. However, in samples stored at room temperature (with or without RPMI), this recognition was only possible for up to 48 h due to pronounced cellular degeneration.

Figure 1

Figure (a) shows Pyknosis and chromatin alterations. Geimsa stain 100× and (b) shows Bacterial colonies grade 2. Geimsa stain 40×

Table 1

Given below shows the detailed morphological changes in the four samples at four different time period

Time duration	Morphological Change Score	Refrigerated	RPMI Refrigeration	Room Temperature	RPMI Room Temperature	P
24 h	0	19	23	9	13	0.006
	1	11	7	21	17
48 h	0	6	4	0	0	0.000
	1	23	25	13	13
	2	1	1	17	17
72 h	1	16	20	1	4	0.000
	2	14	10	13	24
	3	0	0	16	2
1 week	1	0	4	0	0	0.000
	2	17	19	7	5
	3	13	7	23	25

Maximum cellularity was noted at 24 h, which reduced with subsequent time period. [Table 2] Up to 48 h from collection, no significant reduction in cell number was observed in either form of storage although from 72 h, onwards a significant decrement had occurred. Preservation of cellularity was comparable in the sample containingRPMI with refrigeration with those which were simply refrigerated. Samples with RPMI at room temperature had worst cellularity than the normal room temperature samples without preservation. With Fisher's exact test on the four groups, no significant correlation was found at 24 h duration. However, at 48 h, 72 h, and 1-week duration, significance was noted with P value less than 0.05.

Table 2

Cellularity changes in the four samples over different time duration

Time duration	Cellularity score	Refrigerated	RPMI refrigeration	Room temperature	RPMI room temperature	P
24 h	1	30	29	28	27	0.510
	2	0	1	2	3
48 h	1	23	22	0	12	0.000
	2	7	8	29	16
	3	0	0	1	2
72 h	1	6	8	0	2	0.00
	2	22	18	6	13
	3	2	4	21	13
	4	0	0	3	2
1 week	2	10	8	0	0	0.000
	3	20	18	15	12
	4	0	4	15	18

As shown in Table 3, bacterial colonies were more in samples with RPMI at room temperature [Figure 1b] as the RPMI solution acted as nutrient broth for the growth of bacteria at room temperature. The refrigerated samples were slightly less susceptible to bacterial growth. Statistical correlation using Fisher's exact test showed a significant difference in the four samples pointing out RPMI as the main culprit for the same.

Table 3

Bacterial colonies growth in different storage sample over different time duration

Time duration	Bacterial colonies score	Refrigerated	RPMI refrigeration	Room temperature	RPMI room temperature	P
24 h	0	30	29	26	15	0.00
	1	0	1	4	15
48 h	0	30	23	20	2	0.00
	1	0	7	10	20
	2	0	0	0	8
72 h	0	26	7	7	0	0.00
	1	4	23	22	6
	2	0	0	1	24
1 week	0	16	0	1	0	0.00
	1	14	18	19	1
	2	0	12	10	29

DISCUSSION

In the present study, we observed that the cellularity of effusion fluid did not vary significantly in the first 24 h independent of the type of storage. After 48 h, the cellularity reduced from more than 75% to 50%–75% in samples kept at room temperature; however, refrigerated samples with and without RPMI had better cellularity. With regard to morphological cellular analysis, the samples maintained at room temperature had nuclear and cytoplasmatic changes earlier, from 48 h onwards, whereas in samples stored under refrigeration, cell preservation permitted good evaluation for up to 72 h after collection.

Renshaw et al.[6] in their study on cerebrospinal fluid analyzed that specimens with RPMI added preserved at least 90% of their cellularity. In contrast, specimens that were processed without RPMI lost 70% and 94% of their cellularity at 24 h and 48, respectively, even with refrigeration. Compared to our study, the cellularity was reduced by 25% only after 48 h. Cellularity of CSF fluid is much low as compared to those of pleural and peritoneal fluid, and cells in the CSF fluid degenerate more rapidly than the other effusion fluid.

Manosca et al.[8] analyzed 10 samples of cavity fluid for the effect of prolonged storage (up to 14 days) on cell cytometry and morphology. The authors reported there was a distinct reduction in cell numbers over time and also morphological changes that included the presence of blebs and cytoplasmatic vacuolation, generally from 72 h on, although more profuse around day 7. Similarly, in our study, major morphological changes were noted after 72 h onwards.

L Antonangelo et al.[7] evaluated 30 pleural fluids stored at different temperatures i.e., room temperature and refrigerated. He noted that depending on the storage conditions, the samples had quantitative cellular variations from day 3 or 4 onwards. They described that the samples maintained at room temperature had morphological alterations that occurred earlier than in those under refrigeration (day 4) which was identical to our study.

Conner et al.[9] evaluated the variation in leucocyte numbers in pleural fluid determined at 4 and 24 h after thoracocentesis in samples collected in tubes with different characteristics and that were processed manually or automatically. Independent of the tube used, the authors observed no significant variations in the total or differential counts of leucocytes in the samples stored under refrigeration for up to 24 h after collection. As in our study, no major changes occured in any of the samples independent of the storage or preservative used.

There was an overall increase in the risk of contamination of the RPMI as it acted as nutrient broth. The bacterial growth in the RPMI mixed sample at room temperature was many-fold higher than that of refrigerated. The bacterial colonies obscured the morphology of the cell and further added to the degeneration of the cells. After 48 h, maximum bacterial growth was seen in all the samples except those stored in the refrigerator.

CONCLUSION

RPMI medium can be only used along with refrigeration, and good preservative is equivalent to only refrigeration. Therefore, we recommend not using RPMI media as this increases the overall cost of processing.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.