Lymphopenia as a diagnostic biomarker in clinical COVID-19: insights from a comprehensive study on SARS-CoV-2 variants

Linfopenia como biomarcador diagnóstico na COVID-19 clínica: evidências de um estudo abrangente sobre variantes do SARS-CoV-2

Abstract

The enduring SARS-CoV-2 pandemic necessitates robust tools for severity assessment. This study, conducted at Islamabad Diagnostic Center across Pakistan from January 2021 to August 2022, aimed to investigate hematological abnormalities among suspected SARS-CoV-2 subjects. Initial enrollment included 130,347 cases, with 53,078 confirmed positive and 77,269 negative. An additional 11,786 samples expanded the dataset to 142,133. The Omicron and Centaurus variants, in confirmed positive patients, exhibited a slightly higher frequency of hematological abnormalities (30.42%) than negative participants (27.01%). Notably, lymphocyte count reduction (40.95%) suggested its potential as an alternative diagnostic parameter for clinical COVID-19. Decreased levels of NA (37.99%), HGB (26.17%), MCV (20.60%), PLT (6.15%), and ALB (2.28%) were observed. Abnormally elevated NEU, CR, MONO, RBCs, WBC, and EOS levels affected 26.00%, 24.28%, 30.79%, 22.02%, 6.28%, and 5.53% of subjects, respectively. Comparatively, positive patients exhibited higher abnormal blood parameters—LYMP count (57.40%), NEU count (46.08%), EOS count (62.48%), MONO count (31.61%), RBC count (30.32%), ALC count (43.60%), CR count (30.91%), NA count (40.53%), CRP count (68.46%), and DD (63.08%) than negative counterparts. The study underscores lymphocytopenia's potential as a cost-effective, early diagnostic biomarker for clinical COVID-19, preceding real-time PCR diagnosis. This supports its consideration in resource-limited settings for strategic screening and policy-making in the ongoing SARS-CoV-2 battle.

Keywords:
SARS-CoV-2; hematological abnormalities; lymphocytopenia; COVID-19 severity; diagnostic biomarkers

Resumo

A pandemia duradoura do SARS-CoV-2 exige ferramentas robustas para avaliação da gravidade clínica. Este estudo, realizado no Islamabad Diagnostic Center em todo o Paquistão, de janeiro de 2021 a agosto de 2022, teve como objetivo investigar anormalidades hematológicas em indivíduos suspeitos de infecção por SARS-CoV-2. A triagem inicial incluiu 130.347 casos, com 53.078 positivos confirmados e 77.269 negativos. Um acréscimo de 11.786 amostras expandiu o conjunto de dados para 142.133. As variantes Ômicron e Centaurus, em pacientes positivos confirmados, exibiram uma frequência ligeiramente maior de anormalidades hematológicas (30,42%) em comparação com os participantes negativos (27,01%). Notavelmente, a redução da contagem de linfócitos (40,95%) sugeriu seu potencial como parâmetro diagnóstico alternativo para COVID-19 clínica. Foram observados níveis reduzidos de sódio (NA – 37,99%), hemoglobina (HGB – 26,17%), volume corpuscular médio (MCV – 20,60%), plaquetas (PLT – 6,15%) e albumina (ALB – 2,28%). Níveis anormalmente elevados de neutrófilos (NEU), creatinina (CR), Monócitos (MONO), hemácias (RBCs), leucócitos totais (WBC) e eosinófilos (EOS) afetaram 26,00%, 24,28%, 30,79%, 22,02%, 6,28% e 5,53% dos indivíduos, respectivamente. Comparativamente, os pacientes positivos exibiram parâmetros sanguíneos anormais mais elevados – contagem de linfócitos (LYMP – 57,40%), contagem de neutrófilos (NEU – 46,08%), contagem de eosinófilos (EOS – 62,48%), contagem de monócitos (MONO – 31,61%), contagem de hemácias (RBC – 30,32%), contagem de absoluta de linfócitos (ALC – 43,60%), contagem de creatinina (CR – 30,91%), contagem de sódio (NA – 40,53%), contagem de proteína C-reativa (CRP – 68,46%) e dímero (DD – 63,08%), em comparação aos pacientes negativos. O estudo ressalta o potencial da linfocitopenia como biomarcador de diagnóstico precoce e econômico para a COVID-19 clínica, precedendo o diagnóstico por PCR em tempo real. Essas descobertas reforçam sua aplicabilidade em contextos com recursos limitados, contribuindo para o rastreamento estratégico e formulação de políticas de saúde no enfrentamento contínuo do SARS-CoV-2.

Palavras-chave:
SARS-CoV-2; anormalidades hematológicas; linfocitopenia; gravidade da COVID-19; biomarcadores diagnósticos

1. Introduction

SARS-CoV-2, initially identified as a global public health emergency by the World Health Organization (WHO) in December 2019, has since evolved into an enduring pandemic that has reshaped societies and healthcare systems worldwide (WHO, 2022). As we reflect on the past approximately four years of dealing with this novel coronavirus, the multifaceted challenges and complexities associated with its management have become increasingly apparent. The availability of COVID-19 vaccines marked a pivotal moment in our collective effort to curb the spread of the virus and mitigate its impact on public health (WHO, 2020). However, despite significant strides in vaccination campaigns, the global landscape of SARS-CoV-2 cases remains dynamic and continues to evolve. Ongoing challenges in the vaccine supply chain at a global scale and the emergence of new viral variants have posed persistent hurdles in achieving widespread immunity and control (Worldometer, 2022).

The global count of reported SARS-CoV-2 cases, now exceeding 605 million, serves as a stark reminder of the virus's resilience and adaptability. Among the latest challenges are the Omicron and Centaurus variants, characterized by distinctive symptoms such as fever, body aches, cough, and flu-like manifestations (Saeed et al., 2021a). These variants, while sharing commonalities with earlier strains, have introduced new dimensions of severity, potentially leading to conditions like pneumonia, respiratory distress, acute respiratory distress syndrome, and other severe complications (Pakistan, 2022).

Within the context of Pakistan, where the number of SARS-CoV-2-related infections has surpassed 1.5 million cases, the impact of the virus remains a pressing concern. Despite commendable efforts in vaccinating over 130 million individuals, the specter of rapidly evolving SARS-CoV-2 variants challenges the full protective efficacy of available vaccines (Ruan et al., 2020). This complex interplay between the virus and the human immune response underscores the need for ongoing research and adaptive strategies in our fight against COVID-19. Studies conducted in various parts of the world, including China, have shed light on potential markers of disease severity. Notably, lower lymphocyte counts have been observed in deceased SARS-CoV-2 patients, prompting a deeper exploration of biomarkers such as lymphocyte count to better understand and predict the trajectory of the disease (Lin et al., 2020). As we navigate the intricate web of challenges posed by SARS-CoV-2, it is evident that our journey is far from over. The collaborative pursuit of scientific knowledge, vaccination efforts, and adaptive public health measures remains crucial in our ongoing battle against this persistent global health crisis. The coming years will likely witness further refinements in our understanding of the virus, improvements in treatment modalities, and continued efforts to enhance global vaccination coverage, all with the ultimate goal of bringing an end to the devastating impact of SARS-CoV-2 on our societies.

2. Material and Methods

A cross-sectional study was conducted at Islamabad Diagnostic Center, encompassing over 100 branches across Pakistan, on COVID-19 patients from January 2021 to August 2022. The primary objective was to investigate the complete blood profile among suspected SARS-CoV-2 subjects. Trained phlebotomists and counselors performed pre-test counseling, ensuring that all enrolled participants were adequately informed before the examination, and their consent was obtained. Nasopharyngeal swab samples and blood specimens were collected from all suspected clinical COVID-19 Omicron and Centaurus patients presenting with fever and/or respiratory symptoms. The nasopharyngeal swab samples were subjected to SARS-CoV-2 examination via real-time PCR after RNA extraction using Auto pure 32 Zybio, China. Following the recommendations of the World Health Organization (WHO), the USFDA-approved triple target gene (SARS-CoV-2 RNA-dependent RNA polymerase gene, N gene, and Sarbecovirus E gene) based detection Allplex 2019-nCoV Assay (Seegene, South Korea) was utilized. Cycle threshold values below 40 were considered indicative of SARS-CoV-2 positivity. Hematological alterations were assessed for LYMP, MCH, NEU, HCT, AMC, EOS, MCHC, MONO, HGB, RBC, PLT, WBC, RDW-CV, EOS, ANC, ALC, MCV, CR, ALB, NA, FER, CRP, and DD tests using the Sysmex XN-9000 automated analyzer (Sysmex, Kobe, Japan) based on a flow cytometer employing the impedance principle linked to an optical system via a laser-emitted beam of light.

3. Results

A total of 130,347 suspected Omicron and Centaurus SARS-CoV-2 variants were initially enrolled for the study. Out of these, 53,078 individuals were confirmed as SARS-CoV-2 positive (detected via real-time PCR), and 77,269 individuals tested negative. To augment the dataset, an additional 11,786 samples were included, resulting in a revised total of 142,133 samples. Among the confirmed SARS-CoV-2 positive patients with Omicron and Centaurus variants, 57.17% were males, and 42.83% were females.

In comparison to SARS-CoV-2 negative participants with altered hematological profiles (27.01%), the SARS-CoV-2 Centaurus and Omicron positive patients exhibited a slightly higher frequency of hematological abnormalities (30.42%). Notably, among SARS-CoV-2 infected individuals, slightly reduced levels of LYMP (40.95%) were observed, suggesting the potential of lymphocyte count as an alternative clinical diagnostic parameter for clinical COVID-19 patients. Furthermore, NA (37.99%), HGB (26.17%), and MCV (20.60%), PLT (6.15%), and ALB (2.28%) levels were reduced. Abnormally raised levels of NEU, CR, MONO, RBCs, WBC, and EOS were found among 26.00%, 24.28%, 30.79%, 22.02%, 6.28%, and 5.53% of subjects, respectively.

Among SARS-CoV-2 positive patients, in contrast to SARS-CoV-2 negative patients, abnormal blood parameters count included LYMP count among 57.40% (in comparison to 51.30%), abnormal NEU count was found among 46.08% (in comparison to 40.10%), abnormal EOS count among 62.48% (compared to 37.88% of SARS-CoV-2 Neg), abnormal MONO count in 31.61% (than 19.32% of SARS-CoV-2 Neg), abnormal RBC count in 30.32% (than 29.74%), abnormal ALC count among 43.60% (than 29.88%), abnormal CR count among 30.91% (than 26.40%), abnormal NA count among 40.53% (than 27.00%), abnormal CRP count among 68.46% (than 61.80%), abnormal DD among 63.08% (than 57.24%) individuals.

4. Discussion

The severity of SARS-CoV-2 infection is often assessed through various biological markers, including lymphopenia, platelet count, D-dimer, and IL-6, which have been studied extensively in the context of beta-CoV infections such as SARS-CoV and MERS-CoV (Lin et al., 2020). Similar to these previous infections, a significant decrease in CD4+ and CD8+ lymphocytes is observed in individuals infected with SARS-CoV-2, a phenomenon potentially attributed to lymphocyte sequestration at distinct target organ sites (Lin et al., 2020; Li et al., 2004).

Structurally, SARS-CoV is akin to MERS-CoV, both utilizing different receptors during cell entry. SARS-CoV binds to angiotensin-converting enzyme 2 (ACE2), while MERS-CoV binds to cellular dipeptidyl peptidase 4 (DPP4) for cell entry (Raj et al., 2013; Xu et al., 2020). The current study sheds light on the decrease in lymphocyte count among suspected clinical SARS-CoV-2 patients exhibiting symptoms such as fever, cough, running nose, breathing discomfort, flu-like symptoms, and others. However, the precise mechanism behind the reduction in lymphocytes during SARS-CoV-2 infection remains poorly understood. It is posited that ACE2 receptor-expressing lymphocytes may serve as a facilitator for SARS-CoV-2 infection, and subsequent release of pro-inflammatory cytokines, such as Interleukin 6, may contribute to the downregulation of lymphocyte levels (Lin et al., 2020; Xu et al., 2020).

Our findings align with previous investigations during the first three waves of COVID-19 in Pakistan, where several biomarkers and high-resolution tomography results were evaluated for suspected SARS-CoV-2 patients infected with variants like B.1.1.7, delta, and epsilon (Saeed et al., 2021a). In the current study focusing on the prevalent Omicron variant of SARS-CoV-2, we observed a significant increase in levels of Ferritin (FER) by 47.67% and C-reactive protein (CRP) by 70.46%. These results underscore the importance of monitoring these biomarkers in assessing the severity and progression of SARS-CoV-2 infections. The continual rise in viral infections highlights the pressing need for reliable diagnostic methods to mitigate the future burden of diseases (Nawaz et al., 2025a, b; Uppal et al., 2025; Khan et al., 2025; Saeed et al., 2014, 2015, 2021b, c, d, e, f, 2022; Saeed and Manzoor, 2014; Uppal et al., 2022).

5. Conclusion

Our study underscores the significance of lymphocytopenia, among other diagnostic parameters, as a well-established biomarker for assessing the severity of COVID-19. Notably, we present novel findings demonstrating that lymphocytopenia can serve as a valuable and cost-effective diagnostic biomarker specifically for clinical Omicron and Centaurus SARS-CoV-2 patients exhibiting symptoms such as fever, body aches, cough, respiratory discomfort, flu-like symptoms, and others, even before the utilization of real-time PCR-based testing. This observation highlights the potential for early identification and intervention, contributing to more efficient management and control of SARS-CoV-2 infections, particularly in regions where the Omicron and Centaurus variants are prevalent. Further research and validation are warranted to solidify the utility of lymphocytopenia as a frontline diagnostic tool in the ongoing battle against the evolving landscape of COVID-19 variants.

Data Availability Statement

The data is available and can be used for the academic or research purposes.

References

• KHAN, M.A., ASIF, R., AFZAL, R., KHALID, A., SHAHBAZ, S., MUHAMMAD, B., QURESHI, U.J. and GILANI, S.S., 2025 [viewed 11 March 2024]. Environmental parameters and beyond: exploring salts, oils, plant extracts, and honey against COVID-19. Proceedings of the National Academy of Sciences of the United States of America [online], vol. 1, no. 1, pp. 29-39. Available from: https://www.pnas.co.uk/index.php/pnas/article/view/11
  » https://www.pnas.co.uk/index.php/pnas/article/view/11
• LI, T., QIU, Z., ZHANG, L., HAN, Y., HE, W., LIU, Z., MA, X., FAN, H., LU, W., XIE, J., WANG, H., DENG, G. and WANG, A., 2004. Significant changes of peripheral T lymphocyte subsets in patients with severe acute respiratory syndrome. The Journal of Infectious Diseases, vol. 189, no. 4, pp. 648-651. http://doi.org/10.1086/381535 PMid:14767818.
  » http://doi.org/10.1086/381535
• LIN, L., LU, L., CAO, W. and LI, T., 2020. Hypothesis for potential pathogenesis of SARS-CoV-2 infection: a review of immune changes in patients with viral pneumonia. Emerging Microbes & Infections, vol. 9, no. 1, pp. 727-732. http://doi.org/10.1080/22221751.2020.1746199 PMid:32196410.
  » http://doi.org/10.1080/22221751.2020.1746199
• NAWAZ, M., TAHIR, H.S., FATIMA, M., KHALID, M., SAEED, M., ARSHED, Z.Z., SHAHZAD, M., RAJPOOT, F.S., UPPAL, R. and UPPAL, M.R., 2025a [viewed 11 March 2024]. Prevalence of Hepatitis C Virus (HCV) across age and gender in a sample population: a cross-sectional study in Pakistan. Proceedings of the National Academy of Sciences of the United States of America [online], vol. 1, no. 1, pp. 1-9. Available from: https://www.pnas.co.uk/index.php/pnas/article/view/8
  » https://www.pnas.co.uk/index.php/pnas/article/view/8
• NAWAZ, M., FATIMA, M., FAKHAR, M., BUTT, M.F.F., AKMAL, M., NOOR, A., SHAKEYL, M.S., JAWAID, N., ISAAD, F., UPPAL, R. and UPPAL, M.R., 2025b [viewed 11 March 2024]. Epidemiological analysis of dengue fever: insights from demographic, serological, and temporal data in Pakistan. Proceedings of the National Academy of Sciences of the United States of America [online], vol. 1, no. 1, pp. 10-18. Available from: https://www.pnas.co.uk/index.php/pnas/article/view/9
  » https://www.pnas.co.uk/index.php/pnas/article/view/9
• PAKISTAN, 2022 [viewed 11 March 2024]. COVID [online]. Available from: https://covid.gov.pk/
  » https://covid.gov.pk/
• RAJ, V.S., MOU, H., SMITS, S.L., DEKKERS, D.H.W., MÜLLER, M.A., DIJKMAN, R., MUTH, D., DEMMERS, J.A.A., ZAKI, A., FOUCHIER, R.A.M., THIEL, V., DROSTEN, C., ROTTIER, P.J.M., OSTERHAUS, A.D.M.E., BOSCH, B.J. and HAAGMANS, B.L., 2013. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature, vol. 495, no. 7440, pp. 251-254. http://doi.org/10.1038/nature12005 PMid:23486063.
  » http://doi.org/10.1038/nature12005
• RUAN, Q., YANG, K., WANG, W., JIANG, L. and SONG, J., 2020. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Medicine, vol. 46, no. 5, pp. 846-848. http://doi.org/10.1007/s00134-020-05991-x PMid:32125452.
  » http://doi.org/10.1007/s00134-020-05991-x
• SAEED, U. and MANZOOR, S., 2014. Risk factors associated with transmission of hepatitis B and hepatitis C virus in Pakistan. Global Journal of Medical Research, vol. 14, no. 1, pp. 14-19.
• SAEED, U., WAHEED, Y. and ASHRAF, M., 2014. Hepatitis B and hepatitis C viruses: a review of viral genomes, viral induced host immune responses, genotypic distributions and worldwide epidemiology. Asian Pacific Journal of Tropical Disease, vol. 4, no. 2, pp. 88-96. http://doi.org/10.1016/S2222-1808(14)60322-4
  » http://doi.org/10.1016/S2222-1808(14)60322-4
• SAEED, U., WAHEED, Y., ASHRAF, M., WAHEED, U., ANJUM, S. and AFZAL, M.S., 2015. Estimation of hepatitis B virus, hepatitis C virus, and different clinical parameters in the thalassemic population of capital twin cities of Pakistan. Virology : Research and Treatment, vol. 6, VRT.S31744. http://doi.org/10.4137/VRT.S31744 PMid:26568681.
  » http://doi.org/10.4137/VRT.S31744
• SAEED, U., UPPAL, S.R., PIRACHA, Z.Z., RASHEED, A., AFTAB, Z., ZAHEER, H. and UPPAL, R., 2021a. Evaluation of SARS-CoV-2 antigen-based rapid diagnostic kits in Pakistan: formulation of COVID-19 national testing strategy. Virology Journal, vol. 18, no. 1, pp. 34. http://doi.org/10.1186/s12985-021-01505-3. PMid:33581714.
• SAEED, U., UPPAL, S.R., PIRACHA, Z.Z. and UPPAL, R., 2021b. COVID-19 transmission via fomites at low temperature: a potential silent SARS-CoV-2 propagation route. American Journal of Biomedical Science and Research, vol. 12, no. 1, pp. 80-82.
• SAEED, U., UPPAL, S.R., PIRACHA, Z.Z., KHAN, A.A., RASHEED, A., ZAHEER, H., AFTAB, Z. and UPPAL, R., 2021c. Effectivity analysis of SARS-CoV-2 nasopharyngeal swab rapid testing kits in Pakistan: a scenario of inadequate COVID-19 diagnosis. Research Square http://doi.org/10.21203/rs.3.rs-315851/v1
  » http://doi.org/10.21203/rs.3.rs-315851/v1
• SAEED, U., RIZWAN UPPAL, S., ZAHID PIRACHA, Z. and UPPAL, R., 2021d. Azithromycin treatment for SARS-CoV-2-related COVID-19 pandemic could worsen extensively drug resistant (XDR) typhoid: a risk of losing the last bullet against Salmonella enterica Serovar Typhi Jundishapur Journal of Microbiology, vol. 14, no. 1, e113874. http://doi.org/10.5812/jjm.113874
  » http://doi.org/10.5812/jjm.113874
• SAEED, U., UPPAL, S.R., PIRACHA, Z.Z. and UPPAL, R., 2021e. SARS-CoV-2 spike antibody levels trend among sinopharm vaccinated people. Iranian Journal of Public Health, vol. 50, no. 7, pp. 1486-1487. http://doi.org/10.18502/ijph.v50i7.6640 PMid:34568189.
  » http://doi.org/10.18502/ijph.v50i7.6640
• SAEED, U., UPPAL, S.R., PIRACHA, Z.Z., KHAN, A.A., RASHEED, A., WAHEED, A., UPPAL, R. and UPPAL, R., 2021f. Evaluation of SARS-CoV-2 spike antibody levels among Sputnik V first dose vaccinated people in Pakistan: formulation of national anti COVID-19 mass vaccination strategy. Research Square In press. http://doi.org/10.21203/rs.3.rs-480406/v1
  » http://doi.org/10.21203/rs.3.rs-480406/v1
• SAEED, U., PIRACHA, Z.Z., UPPAL, R. and UPPAL, R., 2022. SARS-CoV-2-associated CRP, DD, FER, HBA1c, IL6, LDH, PBNP, and PCT biomarkers and high-resolution computed tomography during the first three waves of COVID-19 in Pakistan (2019-2021). Jundishapur Journal of Microbiology, vol. 15, no. 1, e119590. http://doi.org/10.5812/jjm.119590
  » http://doi.org/10.5812/jjm.119590
• UPPAL, S.R., KHAN, A.A., SAEED, U., PIRACHA, Z.Z. and ALI, Z., 2022. Dermatomycoses in Pakistan; an urgent need for national surveillance programs. Annals of Dermatological Research, vol. 6, no. 1, pp. 14-16. http://doi.org/10.29328/journal.adr.1001021
  » http://doi.org/10.29328/journal.adr.1001021
• UPPAL, R., UPPAL, S.R., UPPAL, M.R., UPPAL, M.S., KHAN, A.A. and MALIK, B.A., 2025 [viewed 11 March 2024]. Harnessing multi-vitamin power to fortify immune defense against SARS-CoV-2: an evidence-based review. Proceedings of the National Academy of Sciences of the United States of America [online], vol. 1, no. 1, pp. 19-28. Available from: https://www.pnas.co.uk/index.php/pnas/article/view/12
  » https://www.pnas.co.uk/index.php/pnas/article/view/12
• WORLD HEALTH ORGANIZATION – WHO, 2020. Coronavirus disease 2019 (COVID-19) situation report. Geneva: WHO, no. 79.
• WORLD HEALTH ORGANIZATION – WHO, 2022 [viewed 11 March 2024]. Coronavirus disease (COVID-19) outbreak [online]. Geneva: WHO. Available from: https://www.who.int/westernpacific/emergencies/covid-19
  » https://www.who.int/westernpacific/emergencies/covid-19
• WORLDOMETER, 2022 [viewed 11 March 2024]. COVID-19 coronavirus pandemic [online]. Available from: www.woldometers.info/coronavirus/
  » www.woldometers.info/coronavirus/
• XU, H., ZHONG, L., DENG, J., PENG, J., DAN, H., ZENG, X., LI, T. and CHEN, Q., 2020. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. International Journal of Oral Science, vol. 12, no. 1, pp. 8. http://doi.org/10.1038/s41368-020-0074-x PMid:32094336.
  » http://doi.org/10.1038/s41368-020-0074-x

Edited by

• Editor:
  Marcelo A.M. Esquisatto

Publication Dates

• Publication in this collection
  22 Sept 2025
• Date of issue
  2025

History

• Received
  11 Mar 2023
• Accepted
  16 Feb 2024