SARS‐CoV‐2 pneumonia and atypical lymphocyte morphology in pleural fluid

CONFLICTS OF INTEREST

Authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

Anna Merino: designed the study and wrote the manuscript. Javier Laguna and Angel Molina: collected and analysed the data. Alexandru Vlagea: performed the immunophenotypic study. Oriol Sibila: performed the clinical evaluation and patient management.

Dear Editors,

Atypical or reactive lymphocytes circulating in peripheral blood of some patients with COVID‐19 infection (hereinafter named COVID‐19 RL) have been reported in previous publications. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ In a recent publication, these lymphocytes were correlated with the evolution and prognosis of the disease. ² In this letter, we present the case of a patient with COVID‐19 infection in which a pneumonia complicated with parapneumonic effusion was also demonstrated. Morphological characteristics of the COVID‐19 RL found in the pleural fluid are described.

A 76 year‐old woman with diabetes mellitus and stage IV breast carcinoma (diagnosed in 2012) was admitted to the Emergency Department of the Hospital for asthenia, vomiting and fever (37.5ºC) in the last two weeks. White blood cell (WBC), red blood cell and platelet counts were normal. Differential WBC count showed high values of neutrophils (9.2 × 10⁹/L) and low values of lymphocytes (0.5 × 10⁹/L). Peripheral blood smear review showed low percentages of normal lymphocytes (5%). Biochemical blood tests showed high values of C‐reactive protein (137 mg/L; normal <10), ferritin (2.06 nmol/L, normal<0.44), lactate dehydrogenase (352 U/L, normal <234) and D‐dimer (3,200 ng/mL; normal <500) were found (see Table 1). Positive RT‐PCR and chest X‐ray examination confirmed the diagnosis of SARS‐CoV‐2 pneumonia, showing the later a right‐sided alveolar consolidation and a large right‐side pleural fluid collection. Two therapeutic thoracenteses were performed with complete drainage and removal of 500 and 700 mL of pleural fluid, respectively. A pleural fluid sample showed a total nucleated cell count of 830 /μL, as is shown in Table 1. After cytocentrifugation and May‐Grünwald‐Giemsa staining of pleural fluid sample, morphology of the cells was analysed. Most of the cells in the differential count corresponded to lymphocytes (74%). Among them, COVID‐19 RL were the predominant (85%), showing a large‐medium size, regular or kidney‐shaped nucleus with a spongy chromatin pattern, usually with one nucleolus, with a distinct moderate to deep blue cytoplasm and occasional presence of small vacuoles. Nucleus showed occasionally an eccentric position (see Figure 1). Among all lymphoid cells, small lymphocytes were 10% and large granular lymphocytes were 6%. Malignant cells were absent.

TABLE 1.

Relevant laboratory parameters in pleural fluid and blood

	Result	Normal values
Pleural fluid results
RBC (/μL)	270	‐
Nucleated cells (/μL)	830	‐
Neutrophils (%)	24	<1
Lymphocytes (%)	74	18‐36
Mesothelial cells (%)	0	<2
Macrophages (%)	2	64‐80
Glucose (mmol/L)	6.2	2.2‐3.9
Total protein (g/L)	40	<30
LDH (U/L)	1,002	‐
ADA (U/L)	180	<33
Blood cell counts
RBC (x1012/L)	3.90	3.80‐4.80
Haemoglobin (g/L)	121	120‐150
Haematocrit (L/L)	0.37	0.36‐0.46
WBC (x109/L)	10.3	4.0‐11.0
Neutrophils (x109/L)	9.2	2.0‐7.0
Lymphocytes (x109/L)	0.5	0.9‐4.5
Platelets (x109/L)	320	130‐400
Blood biochemical tests
Glucose (mmol/L)	9.0	3.6‐6.1
BUN (mmol/L)	4.6	2.1‐8.9
Creatinine (mmol/L)	0.05	0.03‐0.11
GFR (mL/min/1.73 m2)	>90	>90
LDH (U/L)	352	<234
CRP (mg/L)	137	<10
Procalcitonin (μg/L)	0.12	<0.50
Ferritin (nmol/L)	1.17	0.03‐0.44
Cardiac troponin (ng/L)	5.1	<45.2
Coagulation results
PT (seconds)	14.5	9.9‐13.6
D‐dimer (μg/L)	3,200	<500

In bold, abnormal values.

Abbreviation: ADA, adenosine deaminase; BUN, blood urea nitrogen; CRP, C‐reactive protein; GFR, glomerular filtration rate; LDH, lactate dehydrogenase; PT, prothrombin time; RBC, red blood cells; WBC, white blood cells.

FIGURE 1.

Atypical lymphoid cells found in the pleural fluid. Image obtained by cytocentrifugation and May‐Grünwald‐Giemsa staining (1,000 X)

Immunophenotyping of the pleural fluid lymphoid cells by flow cytometry showed high T cell counts (95%), alongside with low B cell counts (3%) and NK cells (2%). Within the T cell population, CD4+T cells were predominant (81% CD4+ T cells vs. 14.1% CD8+ T cells) and TCR expression showed almost exclusively TCRαβ+ (99.7%). Effector memory T cells represented half of the CD4+ T cells, while 50% of CD8+T cells were effector memory and TEMRA T cells, with similar activation levels within both populations. Comparing with cytological and immunophenotyping findings in the case presented herein, in a previous publication analysing a total of 20 Epstein‐Barr virus (EBV) positive pleural effusions, ⁷ lymphocytosis along with a more polymorphous lymphoid population was found, only four cases showed RL (20%), being T cells 2% ‐ 86%, B cells <0.1% ‐ 15% and NK cells <1% ‐ 16%.

Treatment with hydroxychloroquine, azithromycin, lopinavir/ritonavir and antibiotics (ceftriaxone and teicoplanin) was started. In addition, the patient received a single dose of tocilizumab. No recurrence of pleural effusion was observed and a favourable evolution of the infection was observed over the following days. Nevertheless, two months later she presented a progressive dysphagia and a metastasis of the breast carcinoma in the middle third of the oesophagus was detected by endoscopic biopsy, dying after a short period of time.

As was reported by Weinberg et al, atypical lymphocytes in SARS‐CoV‐2 infection are likely reactive to the virus and, in contrast to the classical Downey type II reactive lymphocytes commonly seen in other viral infections such as Epstein‐Barr virus, the morphology of these COVID‐19 RL shows larger size, with a nucleolus and abundant deeply basophilic cytoplasm. ¹ , ² , ⁵ To our knowledge, this is the first morphological description of COVID‐19 RL in the pleural fluid of a patient with SARS‐CoV‐2 pneumonia complicated with parapneumonic effusion.

Literature review reveals that prevalence of pleural effusion can be as high as 14% related to SARS‐CoV‐2 pneumonia. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² In a previous work, the incidence of pleural effusion in patients with acute viral infection increased from 8% incidence to 18% when pleural ultrasonography was used as a screening tool. ¹³ It would be convenient to discard a parapneumonic effusion in patients with suspicion of SARS‐CoV‐2 pneumonia through imaging techniques complementary to chest X‐ray to improve their management.

Pleural fluid analysis in this patient showed an exudative effusion with high lymphocyte count. In contrast with the low percentages of COVID‐19 RL found circulating in peripheral blood, ¹ , ² these lymphocytes were the predominant cells in the pleural fluid of the case presented herein. Previous immunophenotypic studies revealed that presence of COVID‐19 RL suggests an abundant production of virus‐specific T cells, thus explaining the better outcome of patients showing these cells circulating in blood. ² Although our patient showed a good evolution of the infection, she died because of the immediate progression of her malignant disease.

DATA AVAILABILITY STATEMENT

Data available on request from the authors.