377 Journal of Rawalpindi Medical College (JRMC); 2022; 26(3): 377-381 Original Article Reticulocyte Count and Platelet Count as Predictors of Morphological Remission/Hemopoitic Recovery in Acute Lymphoblastic Leukemia (ALL) after Induction Chemotherapy Huma Riaz1, Hamzullah Khan2, Shahtaj Khan3, Sadaf Chiragh4 1 Assistant Professor Hematology, MTI Hayatabad Medical Complex, Peshawar 2 Professor Hematology, Nowshera Medical College, Nowshera. 3 Professor Hematology, MTI Hayatabad Medical Complex, Peshawar 4 Associate Professor Oncology, MTI Hayatabad Medical Complex, Peshawar Author’s Contribution 1,2 Conception of study 1,2,3,4 Experimentation/Study conduction 1,2 Analysis/Interpretation/Discussion 1,2 Manuscript Writing 3,4 Critical Review 3,4 Facilitation and Material analysis Corresponding Author Dr. Hamzullah Khan, Professor Hematology, Nowshera Medical College, Nowshera Email: hamzakmc@gmail.com Article Processing Received: 05/10/2021 Accepted: 02/09/2022 Cite this Article: Riaz, H., Khan, H., Khan, S., Chiragh, S. Reticulocyte Count and Platelet Count as Predictors of Morphological Remission/Hemopoitic Recovery in Acute Lymphoblastic Leukemia (ALL) after Induction Chemotherapy. Journal of Rawalpindi Medical College. 30 Sep. 2022; 26(3): 377-381. DOI: https://doi.org/10.37939/jrmc.v26i3.1790 Conflict of Interest: Nil Funding Source: Nil Access Online: Abstract Objectives: To determine the predictive values of reticulocyte and platelet count for remission in cases of acute lymphoblastic leukemia after induction therapy. Materials and Methods: This cross-sectional observational study was conducted in the department of hematology, MTI Hayatabad Medical Complex, Peshawar. All cases of ALL referred to the department for remission after taking induction therapy, irrespective of age and gender were included. Relevant information was collected on a predesigned proforma prepared in accordance with the objectives of the study. Results: A total of 84 cases referred for remission were included, 56(66.7%) were males and 28 (33.3%) were females. 50(59.5%) cases were in the age range of 5-18 years. The mean with a standard deviation of the age of patients was 15+ 4 years. 75(89.3%) of the cases were classified into ALL-1) by FAB classification. 50(59.5%) of the referred cases had achieved morphological remission by bone marrow aspiration. There was a statistically significant rise in Platelet count of the remission vs non-remission cases (p-0.001). Again there was a statistically significant difference in the retic count of the cases with remission (p-0.05). We observed a statically significant downhill moderate correlation of retic count with remission (in terms of blast count of BM aspiration) (p-0.04, r:- 0.32). Platelet count also had an inverse significant correlation with remission ( p-0.01, r:-0.37). The diagnostic roles of the peripheral platelet count and retic yielded an area under curves of (0.768 and 0.648 respectively) to predict remission. We observed that the retic count and platelet count have been shown to have strong predictive values for remission in ALL with interaction values of (R= 0.28**, ΔR²=0.02, p=0.08). Similarly, an increase in platelet also has a strong predictive value for remission in ALL cases with interaction values of (R= 0.41**, ΔR²=0.16, p=0.001) Conclusion: In ALL cases of post-induction therapy, The peripheral blood reading for an increase in Retic and platelet count predicts remission with 95% confidence. These values if strictly observed can reduce the frequency of invasive procedures like bone marrow aspiration. Keywords: ALL, Remission, Reticulocyte count, Platelet count. 378 Journal of Rawalpindi Medical College (JRMC); 2022; 26(3): 377-381 Introduction Acute Lymphoblastic Leukemia (ALL) is a malignant hematological disorder due to abnormal proliferation of lymphoid progenitors (Lymphoblasts in the bone marrow, peripheral blood, and also in extra medullary sites. ALL occur predominantly in pediatric ages (80%). Its prognosis is good in early ages while poor when occurs in adults. in the United States, its incidence is 1.6/million.1 Globally the remission rate of up to 90% is reported for ALL in pediatric ages.2 Acute Lymphoblastic Leukemia (ALL) prevalence and incidence studies are rarely reported in Pakistan. Similarly, less is reported to highlight the response of ALL to chemotherapy in our population. A local study covering Sindh province reported 70% remission in pediatric age.3 Regarding remission, literature has reported the clinical significance of hematological markers (reticulocyte count, ANC, and platelet) to predict remission in cases of ALL. A regular increase in trends of using these markers has dramatically decreased the frequencies of invasive procedures like bone marrow aspiration and trephine.4. Furthermore immature reticulocyte count as a fraction of new parameters added in advanced hematology analyzers can predict the hemopoitic remission before other tests become positive after induction therapy in cases of ALL. A study reported that 52% of patients showed retic fraction improvement in follow-up cases much before than improvement in ANC in cases of ALL to document hemopoitic recovery.5 A study from Turkey also concluded that immature reticulocyte fraction detects remission in ALL in pediatric age earlier than ANC.6 Platelet recovery or time to platelet recovery (TPR) has been reported to predict disease-free survival (DFS) and also the overall survival rate (OSR) in ALL. Similarly, TPR can also be used as a prognostic factor in leukemia.7 We could not find literature from Pakistan documenting the predictive roles of these hematological parameters in remission cases of ALL, therefore present study was designed to determine the predictive values of reticulocyte, ANC, and platelet count in remission of acute lymphoblastic leukemia after induction therapy to avoid expensive (PCR, Immuno-histochemistry) and invasive (Bone marrow aspiration and trephine) procedures. Materials and Methods This was an observational study conducted in the department of Hematology / Pathology Hayatabad Medical Complex, Peshawar from January 2020 to March 2021. Using Open-Epi software for sample size calculation, a sample size of 84 was selected to represent the true population based on the anticipated proportion of 70% remission in ALL cases, keeping a 95% confidence level and 6% absolute precision due to feasibility issues.3 A total of 84 diagnosed patients of Acute lymphoblastic leukemia (ALL) undergoing induction of remission were included. The diagnosis in all these patients was based on cytological including Giemsa stained peripheral blood smear, bone marrow aspirate smears, and trephine biopsy, immune-phenotypic and cytogenetic criteria. Patients of all ages and both gender were included. Patients with complications and patients in whom standard treatment protocol could not be observed due to any limitation were excluded from our study. All the patients were subjected to a detailed history and clinical examination. On day 07th of the end of the 4th cycle of induction chemotherapy. 3 ml venous blood was collected into a vacuotainer containing K3 EDTA (tripotassium ethylene diamine tetra acetic acid) and platelet counts were performed within 02 hours of sample collection by five parts Sysmex automated haematology Analyzer (XE-3000). A supravital stain of unfixed red blood cells was done with new methylene blue by mixing 100 ul of the patient's blood with 100ul methylene blue, after incubation for 20 minutes at a temperature of 37 ˚ C, the mixture was remixed and wedge blood smear was performed to obtain reticulocyte count, after which the procedure of bone marrow aspiration and trephine biopsy was performed. All the haematological procedures as well as reporting were performed by experienced hematologists. Data was entered in the SPSS version 25 for analysis. The normality of data distribution was determined by Shapiro wilk test. The quantitative variables (Age, Platelet count, retic count) were presented with Mean + SD. The categorical variables were presented with frequency and percentages. An Independent T-test was used to determine the difference between quantitative variables in remission vs non-remission cases. The Pearson correlation test was used to determine the quantitative correlation between continuous variables. For the diagnostic role of the peripheral platelet count and retic, Receiver operating curve (ROC) was used in SPSS to determine the Area under the curve (AUC) to determine the relationship of clinical sensitivity of different hematological 379 Journal of Rawalpindi Medical College (JRMC); 2022; 26(3): 377-381 indicators (Retic and platelet count) and to predict remission. Results A total of 84 cases referred for remission were included, 56(66.7%) were males, and 28 (33.3%) were females. Out of the total, 50(59.5%) cases were in the age range of 5-18 years. The mean with a standard deviation of the age of patients was 15+4 years. 75(89.3%) of the cases were classified into ALL-1) by FAB classification. 50(59.5%) of the referred cases had achieved morphological remission by bone marrow aspiration. (Table 1) We observed a statistically significant rise in Platelet count of the remission vs non-remission cases (p- 0.001). Again there was a statistically significant difference in the retic count of the cases with remission (p-0.05) using the Student t-test as a test of significance. The difference in absolute Neutrophil count in both categories was not statistically significant. (Table 2) We observed a statically significant downhill moderate correlation (Pearson correlation test) of retic count with remission (in terms of blast count of Bone marrow aspiration) (p-0.04, r:-0.32). Platelet count also had an inverse significant correlation with remission (p-0.01, r:-0.37). (Table 3) The diagnostic roles of the peripheral platelet count and retic yielded an area under curves of (0.768 and 0.648 respectively) to predict remission. (Figure 1) Table 1: Descriptive statistic of patients a. Gender wise distribution of patients Frequency Percent Male 56 66.7 Female 28 33.3 Total 84 100 b. Age wise distribution of patients Frequency Percent <5 years 10 11.9 5.1-18 years 50 59.5 18-39 years 21 25 >39years 3 3.6 Total 84 100 c. Morphological diagnosis Frequency Percent ALL-1 75 89.3 ALL-2 6 7.1 ALL3 3 3.6 Total 84 100 d. Remission status of patients Frequency Percent Yes 50 59.5 No 34 40.5 Total 84 100 Table 2: The impact of platelet and reticulocyte count on remission in Acute Lymphoblastic Leukemia (ALL) Hematological indices Remission status N Mean+SD P-value Platelet count Yes 50 332.1+136 0.001 No 34 207.9+135 Reticulocyte count Yes 50 1.7+1.4 0.049 No 34 1.4+1.2 ANC Yes 50 5255+37 0.067 No 34 3286+58 Table 3: Correlations of reticulocyte count and platelet count with the number of blast cells in remission cases after induction therapy Morphological remission Reticulocyte count Reticulocyte count rs -.312** p 0.004 N 84 Platelet Count rs -.367** .327** p 0.001 0.002 N 84 84 ** Correlation is significant at the 0.01 level 380 Journal of Rawalpindi Medical College (JRMC); 2022; 26(3): 377-381 Figure 1: Discussion Survival in cases of acute lymphoblastic leukemia mainly depends on complete remission. Complete remission can be defined based on certain variables in different procedures/investigations to be more accurately defined remission. However, we hypothesized that a reticulocyte count of more than 2.5% and a platelet count of more than 250000/cmm3 is essential to label a case as remission before going into the invasive procedures. In the present study, the mean age of patients was 15+4 years with male gender predominance (66.7%). 89.3% of the cases were classified into ALL-1 by FAB classification. The correlation strength of retic count and platelet count with remission was (p-0.04, r:-0.32), (p-0.01, r:-0.37) respectively. A study on the bio-clinical and demographic (age & gender) characteristics of a large cohort of 5000 ALL patients concluded that the peak incidence of ALL was reported at 1-5 years of age followed by another spike in age 6-10 years, with a progressive decrease in cohort 11-30 years, and disappear in the 50-60 years cohort. Furthermore, they reported a lower incidence in the female gender which supports our findings.8 Rauf et al6 also reported the mean age of children with ALL as 12+5 years in their study conducted in the department of hematology, Armed force institute of Pathology Rawalpindi which matches the age of occurrence of ALL with our population. The rate of morphological remission was 59.5% in the present study which is lower than as reported by Vallacha A et al7 from Karachi (70%). Another study from malysia5 reported 63% remission which is in concordance with our findings. In European countries, a remission rate of up to 90-95% is reported in pediatric age.9 Regarding the clinical significance of the hematological parameter in remission, a statistically significant rise in Platelet count and retic count was observed in cases with remission vs no remission (p-0.001) and (p-0.05) respectively. While no such difference in absolute Neutrophil count in both the categories was noted in groups. A study published in “Leukemia” journal reported an increase in reticulocyte count in all patients who achieved remission /recovery after chemotherapy (ATCO) (p<0.001) that matches our findings.10 Reticulocyte count is used by many clinical centers to assess hemopoitic recovery in ALL cases. Microscopically quantification is done by hematologists and flowcytometry or advanced hematology analyzers can give you accurate immature reticulocyte fractions (IRF). According to a study performed by Rawalpindi AFIP to show remission success in ALL, they reported 78% sensitivity for IRF 381 Journal of Rawalpindi Medical College (JRMC); 2022; 26(3): 377-381 to predict remission which is higher than the present study.11 Replication analysis has also confirmed the prognostic value of platelet count on treatment day 33 and has a strong inverse correlation with the number of blast cells after induction therapy (P<0.001) 12 which is consistent with our findings. we did not measure and analysed the platelet indices to see the correlation of the individual platelet indices with remission, a study from Bangladesh reported that Platelet count and platelet-crit (PCT) were significantly higher in the post-therapy remission cases versus non-remission cases (p<0.05) 13 still in term of platelet count it correlates with our findings. Total leukocyte count (TLC) and ANC are easily ascertainable hematological parameters that reflect the plasma levels of chemotherapy to achieve remission in ALL. However, we could not appreciate the difference in absolute Neutrophil count in remission cases versus no remission groups and our findings are in concordance with the study of Lucas K et al.14 However they have reported an inverse relationship of post-induction therapy with TLC and ANC. A study reported mean TLC in relapsed and non-relapsed groups respectively (p = 0.03) and the same was for mean ANC (x 10(3)/mm3) were 3.0 +/- 0.9 and 2.5 +/- 0.6 in both groups (p = 0.05).13 Authors have reported ANC with 96% sensitivity even to predict the hemopoitic recovery in ALL.15 On the ROC curve, the platelet count and retic yielded an area under curves of (0.768 and 0.648 respectively) to predict remission. Another study from China reported using the Receiver Operating Curve (ROC) and regression analysis with findings that platelet count can have a predictive value for the prognosis of patients with acute leukemia.16 Conclusion The higher reticulocyte and platelet count in cases of post-induction therapy can predict remission in acute lymphoblastic leukemia (ALL). There is a statistically significant inverse correlation of reticulocyte and platelet count with the number of lymphoblasts in ALL after induction therapy. The diagnostic values of reticulocyte and platelet count on the ROC curve as acceptable to be used in clinical setup to reduce the frequency of invasive procedures like (bone marrow aspiration and trephine biopsy) and expensive procedures (immune histochemistry and PCR). References 1. Terwilliger T, Abdul-Hay M. Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J. 2017 Jun 30;7(6):e577. doi: 10.1038/bcj.2017.53. PMID: 28665419; PMCID: PMC5520400. 2. Riley S, Jonathon M, Ezra B, Goel R, Tidwell A. Reticulocytes and Reticulocyte Enumeration. Journal of Clinical Laboratory Analysis 2001;15:267- 294 3. Vallacha A, Haider G, Raja W, Kumar D. Remission Rate of Acute Lymphoblastic Leukemia (ALL) in Adolescents and Young Adults (AYA). J Coll Physicians Surg Pak. 2018 ;28(2):118-21. 4. Bhatnagar S, Chandra J, Narayan S. Hematological changes and predictors of bone marrow recovery in patients with neutropenic episodes in acute lymphoblastic leukemia. J Trop Pediatr. 2002; 48(4):200-3. doi: 10.1093/tropej/48.4.200. PMID: 12200979. 5. Raja-Sabudin RZ, Othman A, Ahmed-Mohamed KA, Ithnin A, Alauddin H, Alias H, Abdul-Latif Z, Das S, Abdul-Wahid FS, Hussin NH. Immature reticulocyte fraction is an early predictor of bone marrow recovery post-chemotherapy in patients with acute leukemia. Saudi Med J. 2014;35(4):346-9. 6. Rauf SE, Khan SA, Ali N, Afridi NK, Haroon M, Arslan A. Immature Reticulocyte Fraction and Absolute Neutrophil Count as Predictor of Hemopoietic Recovery in Patients with Acute Lymphoblastic Leukemia on Remission Induction Chemotherapy. Turk J Haematol. 2016;33(2):131-4. doi: 10.4274/tjh.2014.0379. 7. Faderl S, Thall PF, Kantarjian HM, Estrov Z. Time to platelet recovery predicts outcome of patients with de novo acute lymphoblastic leukaemia who have achieved a complete remission. Br J Haematol. 2002 ;117(4):869-74. doi: 10.1046/j.1365-2141.2002.03506.x. 8. Foa R. Acute lymphoblastic leukemia: age and biology. Pediatr Rep. 2011 Jun 22;3 Suppl 2(Suppl 2):e2. doi: 10.4081/pr.2011.s2.e2. 9. Redaelli A, Laskin BL, Stephens JM, Botteman MF, Pashos CL. A systematic literature review of the clinical and epidemiological burden of acute lymphoblastic leukaemia (ALL). Eur J Cancer Care (Engl). 2005 Mar;14(1):53-62. doi: 10.1111/j.1365-2354.2005.00513.x. 10. Kajiguchi, T, Yamamoto, K, Sawa, M. Increased erythropoietin level and reticulocyte count during arsenic trioxide therapy. Leukemia 2005;19:1–3 https://doi.org/10.1038/sj.leu.2403635 11. Rehman H, Zafar L, Rehan M, Khalique S, Imran, Imran T. Post Chemotherapy Bone Marrow Recovery in Acute Leukaemias- Comparison of Immature Reticulocyte Fraction with Absolute Neutrophil Count. Jour Rawalpind Med coll, 2015; 19(2):117-119 12. Zeidler L, Zimmermann M, Moricke A, Meissner B, Bartels D, Tschan C, Schrauder A, Cario G, et al. Low platelet counts after induction therapy for childhood acute lymphoblastic leukemia are strongly associated with poor early response to treatment as measured by minimal residual disease and are prognostic for treatment outcome. Haematologica. 2012; 97(3):402-9. 13. Khan M, Morshed AA, Ahmed TU, Khan HH, Ahmed AU, Roy S, et al. Platelet Indices As Markers For Remission In ALL During Induction of Remission: An Experience of 52 Cases. BANGLADESH J CHILD HEALTH 2020; VOL 44 (1) : 34-39 14. Lucas K, Gula MJ, Blatt J.Relapse in acute lymphoblastic leukemia as a function of white blood cell and absolute neutrophil counts during maintenance chemotherapy. Pediatr Hematol Oncol, 1992;9(2):91-7 15. Buttarello M, Bulian P, Farina G, Petris MG, Temporin V. Five fully automated methods for performing immature reticulocyte fraction. Am J ClinPathol 2002;117:871-79 16. Zhang Q, Dai K, Bi L, Jiang S, Han Y, Yu K, Zhang S. Pretreatment platelet count predicts survival outcome of patients with de novo non-M3 acute myeloid leukemia. PeerJ. 2017 Dec 21;5:e4139. doi: 10.7717/peerj.4139.