The clinical value of high fluorescent lymphocytes and smudge cells in the diagnosis of infectious mononucleosis

Abstract Background The diagnostic value of high fluorescent lymphocytes (HFLC) and smudge cells in diseases like sepsis has been confirmed. In this study, we explore the diagnostic value of HFLC and smudge cells for infectious mononucleosis (IM). Methods Sixty‐two IM patients, 67 healthy controls, 84 patients with upper respiratory tract virus infection, and 35 patients with malignant lymphoid diseases were enrolled. The complete blood counts and leukocyte differential counts are tested, and the smudge cells were manually counted. Results The value of HFLC% and smudge cells of the IM group were significantly higher than those of healthy controls and disease controls (p < 0.05), and the HFLC% value of IM patients was positively correlated with the number of reactive lymphocytes (r = 0.265). When the cutoff value of HFLC% was 0.4%, and the diagnostic value of IM was high (AUC = 0.995). When the smudge cells >2/100 nucleated cells, it can show better (AUC = 1.000). When the cutoff value of the HFLC% was 1.2%, it can effectively distinguish IM patients from upper respiratory tract virus infection patients (AUC = 0.934); when smudge cells >16/100 nucleated cells, it also has high differential diagnosis value (AUC = 0.913). In addition, the AUC of the combination HFLC% and smudge cells for the differential diagnosis can be increased to 0.968. The performance value of single HFLC% (AUC = 0.942) for distinguishing IM from malignant lymphoid diseases was better than smudge cells and combine index with the cutoff value of 0.4%. Conclusion HFLC% and smudge cells can be used as effective indicators in the early diagnosis and differential diagnosis of IM.


| INTRODUC TI ON
Infectious mononucleosis (IM) is a clinical disease characterized by fever, angina, and lymphadenopathy. It was first proposed by Sprunt and Evans 1 to describe similar acute infections. The disease is accompanied by a syndrome of atypical large lymphocytes in the peripheral blood. This atypical lymphocyte is also called Downey cell. 2 IM is most common in adolescents and young people, and often occurs in late autumn to early spring. It may be caused by a variety of pathogens, and Epstein-Barr virus (EBV) infection accounted for the vast majority of patients. It can be spread through droplets, blood transfusion etc., and humans are generally susceptible. Therefore, IM is also defined as an acutely proliferative self-limiting infectious disease of the mononuclear-macrophage system caused by the EBV.
Although the related clinical symptoms of IM such as fever, fatigue, lymph nodes, and hepatosplenomegaly will gradually disappear within a few months, a small number of patients will still experience splenic rupture, chronic active EBV, hemophagocytic syndrome, and other serious and long-lasting complications. 3 At present, there is no specific treatment method for IM, and there is no approved EBV vaccine. Therefore, early diagnosis of IM, timely medical intervention, to prevent the aggravation of the disease is of great significance.
Clinically, the diagnosis of IM is mainly based on the comprehensive consideration of clinical symptoms, hematology, and serology, and the following criteria are mostly used: ① fever, angina, lymphadenopathy, hepatosplenomegaly, and skin rash; ② increased proportion of lymphocytes, and reactive lymphocytes>10%; ③ heterophilic agglutination test is positive; ④ anti-viral capsid antigen (VCA) IgM antibody is positive; and ⑤the syndrome can be caused by other viruses (cytomegalic virus, etc.) and some bacteria. Atypical lymphocytes appear, but the heterophilic agglutination test is negative. When the patient has three symptoms in ①, meets any two of ②③④, and excludes ⑤, it can be diagnosed as IM. In particular, IM patients who are negative for heterophilic antibodies due to other viral agents are also considered. However, these diagnostic standards have their shortcomings: the clinical manifestations described in ① can appear in a variety of diseases, and the indication of IM is not clear, and it is easy to be misdiagnosed as upper respiratory tract infections and other diseases; and the morphology of reactive lymphocytes is diverse, leading to large differences in judgment among different technicians. 4 The formation of smudge cells leads to serious cell damage and affects the accuracy of manual leukocyte classification. The accuracy of heterophilic agglutination to diagnose IM was 71%-90%; however, the test has a false-negative rate of 25% in the first week of illness, 5 and it is a non-specific test. The high cost of serological antibody testing, long testing time, and short duration of IgM antibodies is prone to miss diagnosis. Therefore, it is very valuable to find novel biomarkers with higher diagnostic efficiency for the diagnosis of IM. In our clinical work, we found that the HFLC% value and the number of smudge cells in patients diagnosed with IM have an increasing trend. The aim of this study was to evaluate the efficiency of HFLC% and smudge cells in the peripheral blood as diagnostic biomarkers of IM.

| Study population
We conducted a prospective observational study in Peking University Third Hospital from August 2020 to February 2021. IM patients were enrolled with the following criteria: patients who met clinically relevant diagnostic criteria mentioned above and were di-

| Statistical analysis
All data analysis was performed using SPSS 24.0 (SPSS, Inc.) and MedCalc 19.6 (MedCalc Software). Continuous variables with nonnormal distribution were shown as median [interquartile range (IQR)], which used non-parametric Mann-Whitney U test to compare between groups. The data with normal distribution were represented as mean ± standard deviation (x±SD), and comparison between groups was performed by t test. The categorical variables were expressed as percentages (%) and compared by chi-square test.
p < 0.05 was considered statistically significant. Spearman's test was used to study the correlation between the HFLC% and the number of reactive lymphocytes in the IM group. The clinical performances of different indicators were assessed by receiver operating characteristics (ROC) curve analysis, the area under the curve (AUC), cutoff values, specificity, and sensitivity were calculated.

| Characteristics of the study population
Basal characteristics and complete blood counts of the study population are summarized in Table 1. There was no significant difference in gender in the four groups, while the patients of IM were significantly younger than that of the control groups. This is consistent with the fact that the prevalence of IM is highest among 15-24 years old. 6 The complete blood counts of the four groups were statistically analyzed. RBC and HGB in IM group were higher than those in the TC group (p < 0.05), but there was no significant difference compared with HC and URIC groups (p > 0.05). WBC and PLT in IM patients were significantly higher than those in HC and URIC group (p < 0.05); however, there was no significant difference compared with those in TC group (p > 0.05). Interestingly, HFLC% was significantly higher in IM patients than in the other three groups (p < 0.05).

| Comparison of the number of smudge cells among the IM group, HC group, URIC group, and TC group
The number of smudge cells in the four groups of study population

| Comparison of leukocyte differentiation between automatic blood analyzer and manual leukocyte classification
We found that there was a significant difference between the per-

| The diagnostic value of HFLC% and the number of smudge cells for IM
The level of HFLC% and the number of smudge cells was significantly higher in patients with IM compared with healthy controls (p < 0.05).
The sensitivity and specificity of smudge cells with the cutoff value of 2 per 100 nucleated cells were 100% ( Figure 5A). a Expressed that the difference between the IM group and the HC group is statistically significant (p < 0.05).

TA B L E 1 Basal characteristics and complete blood counts of the study population
b Expressed that the difference between the IM group and the URIC group is statistically significant (p < 0.05).
c Expressed that the difference between the IM group and the TC group is statistically significant (p < 0.05).

| The value of HFLC% and number of smudge cells in differentiating IM from upper respiratory tract virus infection
The optimal HFLC% was >1.2% with a sensitivity of 85.71%, speci-  Figure 5B).

| The value of HFLC% and the number of smudge cells in differentiating IM from malignant lymphoid diseases
Compared with smudge cells, the value of HFLC% in differentiating IM from malignant lymphoid diseases was much better (p < 0.05). Moreover, combined detection of these two indicators could not further improve the performance (Table 4). When cutoff value of HFLC% was 0.4%, the differential diagnosis value of  The analysis showed that the counts of HFLC in patients infected with dengue fever were significantly higher than that of patients infected with other pathogens (p < 0.001), indicating that HFLC has clinical value in the diagnosis of patients infected with dengue fever. 9 Arneth BM et al. found an early elevation of HFLC in a patient who developed from a urinary tract infection to uremia. They conducted a retrospective study HFLC on 38 patients in intensive care units (ICU) to further research. The results showed that in the group of non-infection patients, the HFLC value was very low, the reference range was less than 0.2%, while in the local infection group, the value increased slightly, and it increased significantly in the patients with sepsis, suggesting that HFLC has auxiliary diagnostic value in sepsis. 11 Our study confirmed that HFLC% has a good clinical diagnostic value in IM patients. In clinical practice, the most common diseases with lymphocyte increase are upper respiratory tract virus infection and malignant lymphoid diseases. Therefore, these two diseases, which are similar to IM in clinical presentation and laboratory examination, were used as control groups in this study to evaluate the value of the novel indicators in the diagnosis and differential diagnosis of IM. HFLC% value of the IM patients was significantly higher than that of the healthy individuals and the disease control groups (p < 0.05). When the cutoff value was 0.4%, IM patients can be identified from healthy controls with a high sensitivity of 98.39%, a high specificity of 97.01%, and an AUC of 0.995 (95% CI 0.963-1.000).
In addition, with this cutoff value, HFLC% can effectively distinguish IM from malignant lymphoid diseases, whose AUC was 0. There are some limitations in this study. First of all, after the SARS-CoV-2 epidemic broke out, people became more aware of virus protection and began to wear masks. As a result, the number of patients with IM who went to the hospital decreased compared with previous years, which resulted in a limited number of samples in this study. To determine the disease-specific cutoff value and reference range, larger and more diverse sample data are necessary to be included. In addition, the influence of disease progression, drug use, lifestyle, age, and other factors on the HFLC% level of patients with IM is still uncertain, and further research is needed.

| CON CLUS ION
HFLC% can be used for auxiliary diagnosis of IM, thereby saving time and reducing cost of smear and manual classification. HFLC% assists the diagnosis of IM with the following advantages. First, this indicator has high specificity and sensitivity for the diagnosis of IM, which can effectively avoid missed diagnosed. Second, HFLC%, as one of the blood routine indicators, can be obtained directly from the automatic blood analyzer. It does not increase additional economic burdens and manual operations. It has better adaptability and superiority and can prompt clinical diagnosis and treatment earlier.
In addition, when the number of smudge cells in the peripheral blood increases, the doctor should be alert to the possibility of IM.

ACK N OWLED G M ENT
These studies were supported by research grants from this work supported by grants from programs of the Natural Science Foundation of China (81800604, 62071011).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.