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Diagnostic value of white blood cell parameters for COVID‐19: Is there a role for HFLC and IG?

Abstract Introduction As the Coronavirus disease 2019 (COVID‐19) pandemic is still ongoing with patients overwhelming healthcare facilities, we aimed to investigate the ability of white blood cell count (WBC) and their subsets, high fluorescence lymphocyte cells (HFLC), immature granulocyte count (IG), and C‐reactive protein (CRP) to aid diagnosis of COVID‐19 during the triage process and as indicators of disease progression to serious and critical condition. Methods We collected clinical and laboratory data of patients, suspected COVID‐19 cases, admitted at the emergency department of University General Hospital of Ioannina (Ioannina, Greece). We selected 197 negative and 368 positive cases, confirmed by polymerase chain reaction test for severe acute respiratory syndrome coronavirus 2. COVID‐19 cases were classified into mild, serious, and critical disease. Receiver operating characteristic curve and binary logistic regression analysis were utilized for assessing the diagnosing ability of biomarkers. Results WBC, neutrophil count (NEUT), and HFLC can discriminate efficiently negative cases from mild and serious COVID‐19, whereas eosinopenia and basopenia are early indicators of the disease. The combined WBC‐HFLC marker is the best diagnostic marker for both mild (sensitivity: 90.6% and specificity: 64.1%) and serious (sensitivity: 90.3% and specificity: 73.4%) disease. CRP and Lymphocyte count are early indicators of progression to serious disease whereas WBC, NEUT, IG, and neutrophil‐to‐lymphocyte ratio are the best indicators of critical disease. Conclusion Lymphopenia is not useful in screening patients with COVID‐19. HFLC is a good diagnostic marker for mild and serious disease either as a single marker or combined with WBC whereas IG is a good indicator of progression to critical disease.

of a hospital are treated as suspected COVID-19 patients. Diagnosis of COVID-19 patients usually relies on RT-PCR real-time polymerase chain reaction (RT-PCR) tests, which can be time-consuming. On the other hand, the availability of rapid PCR testing may be restricted.
Given the possible consequences from the delayed diagnosis and quarantine of SARS-CoV-2-positive patients, triage at the emergency department has become a formidable task. patients. However, most of the reports regarding diagnostic value of hematologic parameters for COVID-19 refer mainly to comparison between the control group and COVID-19 patients [3][4][5][6] whereas few studies have evaluated their performance as diagnostic markers in the emergency room. [7][8][9] On the other hand, there is a great load of data for the utilization of hematologic parameters for the diagnosis of progression to serious or severe disease and their performance as prognostic markers in COVID-19 patients. The most common hematologic parameters derived from CBC with evidenced prognostic value include the following: neutrophil count, 10 lymphocyte count, 11,12 neutrophilto-lymphocyte ratio (NLR), 13,14 and platelet-to-lymphocyte ratio (PLR). 14,15 We sought to determine the performance of parameters of white blood cells and their subpopulations as well as their combinations for the diagnosis of COVID-19 and as indicators of disease progression to serious and critical condition. Since the blood cell parameters may depend on the stage of the disease, we classified patients according to their clinical condition into three groups: patients with mild, serious, and critical disease. In this way, we anticipated to discover early diagnostic markers for COVID-19 disease with the potential to optimize the triage process as well as early indicators of disease progression in order to aid clinicians in the management of patients in need of close monitoring for developing serious and/or critical condition. In addition to the parameters of CBC, we also examined other known markers used as indicators of systematic inflammatory response such as NLR, PLR, and lymphocyte-to-monocyte ratio (LMR) 14 as well as high fluorescence lymphocyte cells (HFLC) and Immature Granulocyte count (IG).
HFLC are lymphoplasmacytoid cells or plasma cells present in the blood of patients as a response of the innate immunity to infectious disease. 16 Their detection is based on their characteristical high fluorescence intensity and their count is reported by modern automated hematology analyzers as part of the full blood count.
HFLC are elevated in COVID-19 patients and are further increased in severe disease. 17 On the other hand, immature granulocytes in the peripheral blood can occur in response to infection, inflammation, or other cause of bone marrow stimulation. Both HFLC and IG have been thoroughly investigated as potential markers of sepsis. 18,19 Creactive protein (CRP), a well-known inflammation and disease progression marker, was also included in the study. 7 In this respect, we explored their potential role in the diagnosis of COVID-19 as standalone markers and in combination with other parameters of the CBC.

| Patients
This is a retrospective case-control study conducted from 14 March 2020 to 6 March 2021, with data collected from patients admitted at the emergency department of University General Hospital of Ioannina (Ioannina, Epirus, Greece). Due to the low prevalence of COVID-19 disease in our country from March to October 2020, we had to extend the time of data collection to March 2021 in order to include as many COVID-19 patients as possible. All patients who presented at the emergency department with fever and/or respiratory symptoms were suspected for COVID-19 infection, and their nasopharyngeal swab specimens were tested for SARS-CoV-2 with real-time polymerase chain reaction (RT-PCR). 197 patients who tested negative in RT-PCR were selected as the control group (negative cases). Negative cases discharged to home were considered as mild negative cases (control 1, n = 103) while negative cases admitted to general ward were classified as serious negative cases (control 2, n = 94).
The clinical evaluation and management of SARS-CoV-2-positive patients were performed according to the Guidelines of the National Institute of Public Health of Greece. 21 COVID-19 patients were classified according to their clinical condition as evaluated at the emergency department into three groups defined as following: mild disease: discharged to home, serious disease: hospitalized in general ward and severe/critical disease: admitted to intensive care unit (ICU). Patients initially admitted to general ward and later transferred to ICU (n = 23) were included in the critical group; CBC on admission to ICU was used in this case.
Only adult patients were included in the study whereas patients with conditions associated with abnormal blood cell counts as hematological malignancies, metastatic bone marrow infiltration by malignancy, receiving chemotherapy or immunosuppressive therapy (n = 13) were excluded from the study. We also excluded patients without CBC on admission (n = 3) as well as patients transferred from another ward (n = 7). A total of 368 COVID-19 cases were included in the study and were classified as having mild (n = 96), serious (n = 215), and critical (n = 57) disease ( Figure 1).

| Data collection and management
Demographic data and clinical symptoms and signs were obtained from electronic medical records. The complete blood count and the extended parameters HFLC and IG on day of admission were measured on Sysmex XN-3100 (Sysmex, Japan). CRP measurements were obtained from the hospital Laboratory Information System (LIS). RT-PCR test was performed on Xpert Xpress SARS-COV-2 (Cepheid AB).

| Study design and statistical analysis
The study of CBC parameters as diagnostic markers of COVID-19 comprised of two parts. In the first part, their ability to discriminate between negative and positive cases was tested separately for mild

| Patients
The basic demographic data and laboratory findings of all groups of patients are summarized in Table 1 (Comorbidities are given in

| Hematologic parameters as diagnostic markers of COVID-19
Initially, we performed ROC curve analysis in order to select the best diagnostic markers among CBC parameters and CRP for mild disease (Table S2). The parameters with AUC>0.7 (WBC, NEUT, BASO, HFLC, and EOS) were included in the multivariable analysis, and the odds ratio (OR) for the odds of having mild COVID-19 disease was calculated by conducting logistic regression ( Table 2). Because of the strong correlation between WBC and NEUT (Pearson r = 0.959, P <.0001) and the high VIF values for WBC and NEUT due to collinearity, the best full model could not include both WBC and NEUT.
Therefore, the best fitting logistic model indicated that WBC and HFLC were independently associated with mild COVID-19 disease.
Due to the low values observed for EOS, BASO, and HFLC, adjusted ORs have been calculated in order for the one-unit change of the predictor to be meaningful (see Table 2). Hence, an increase in 0.01 (×10 9 /L) in the value of HFLC corresponds to 1.655 increase in odds of having mild COVID-19 disease.

| Hematologic parameters as indicators of COVID-19 disease progression
The univariable analysis indicated LYMPH and CRP as good indicators of progression from mild to serious COVID-19 disease (Table S3). Furthermore, multivariable analysis revealed that both markers are independent indicators of progression of mild to serious disease ( Table 4). The logistic model of their combination failed the goodness of fit test (Hosmer and Lemeshow test <0.05).
ROC curve analysis highlighted several parameters for the indication of progression from serious to critical illness (Table S3).   The diagnostic ability of leukocyte and neutrophil count for COVID-19 has been highlighted in several different studies. 6,7,9,25 Indeed, WBC and NEUT were significantly lower in mild and serious disease compared with the negative group and were both independent determinants of COVID-19 disease. WBC and NEUT, both displayed high efficiency in the diagnosis of mild and serious COVID-19 disease either as single or in combination with other parameters of the CBC.

| D ISCUSS I ON
NLR has been proposed as a possible sufficient diagnostic marker for the COVID-19 disease. 6,9,14 In our study, NLR is a diag-

CO N FLI C T O F I NTE R E S T
The authors have no competing interests.

E TH I C A L A PPROVA L
Due to the retrospective and non-interventional nature of the study, informed consent was not required. The study was approved by the review board of University General Hospital of Ioannina (registration number: 10/26-05-2021).

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 from the corresponding author upon reasonable request.