Objectives: To analyze whether leucopenia and lymphopenia a characteristic feature of children with COVID-19 and to find out its association with the disease severity. Methods: This was a descriptive cross-sectional study conducted at The Children’s Hospital Lahore from March 2020 to October 2020. All confirmed cases of COVID-19 infection and post-COVID MIS-C/Kawasaki Disease diagnosed on the basis of RT-PCR and Antibody test respectively were included. Complete blood and differential counts were performed on the day of admission. Results: Out of a total of 83 patients 60 (72%) were diagnosed as COVID-19 and 23 (28%) as post-COVID MIS-C/KD. The mean age of children was 7.0±4.3 years (95%CI: 6.07 - 8.75) with a male preponderance 51 (61%). Twenty (24%) children had an underlying comorbidity and 7 (8%) were surgical cases. Our case fatality rate was 5 (6%) and all children who died had an underlying comorbid condition. In both, COVID and MIS-C/KD the mean leukocyte count was (14.0 ± 12.5 vs 13.6 ± 6.9 x109/L), respectively (p=0.888). The mean lymphocyte count in children with COVID was (39.1 ± 21.4%). Patients with MIS-C/KD showed significantly higher levels of neutrophil count (76.5 ± 15.0%) as compared to children with COVID (52.0 ± 22.1%), absolute lymphocyte count was (5.02±4.81 vs 2.13±0.95 x109/L) in COVID and MIS-C respectively (p=<0.001). In 60 COVID-19 patients, the mean neutrophil lymphocyte ratio (NLR) in mild-moderate and severe-critical group was 2.00 and 5.08 respectively (p=0.009). Conclusion: The blood picture of COVID-19 in children does not show leukopenia. NLR was a prognostic factor to assess the severity in COVID-19 patients. The presence of an underlying comorbid conditions is significant a risk factor for poor outcome. doi: https://doi.org/10.12669/pjms.37.3.3848 How to cite this:Bari A, Ch A, Bano I, Saqlain N. Is leukopenia and lymphopenia a characteristic feature of COVID-19 in children?. Pak J Med Sci. 2021;37(3):---------. doi: https://doi.org/10.12669/pjms.37.3.3848 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

BackgroundCongenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder that was first described in 1920. It is transmitted as an autosomal recessive trait that is characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in a higher number of cases of congenital fibrinogen deficiency in their respective populations. This study focused on the detection of mutations in fibrinogen genes using DNA sequencing and molecular modeling of missense mutations in all three genes [Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG)] in Pakistani patients.MethodsThis descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency were screened for mutations in the Fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study.ResultsTen patients had mutations in FGA followed by three mutations in FGB and three mutations in FGG, respectively. Twelve of these mutations were novel. The missense mutations were predicted to result in a loss of stability because they break ordered regions and cause clashes in the hydrophobic core of the protein.ConclusionsCongenital afibrinogenemia is a rapidly growing problem in regions where consanguinity is frequently practiced. This study illustrates that mutations in FGA are relatively more common in Pakistani patients and molecular modeling of the missense mutations has shown damaging protein structures which has profounding effect on phenotypic bleeding manifestations in these patients.

Introduction Type 3 von Willebrand disease (VWD), a severe autosomal recessive hereditary bleeding disorder, is described by the virtual absence of von Willebrand factor (VWF). In consanguineous populations, for example Pakistan, the disease is reported with a higher incidence rate than the worldwide prevalence. Aims This study aims to characterize molecular pathology and clinical profile of type 3 VWD cohort of Pakistani origin. Methods In total, 48 patients were enrolled in the current study. Initially, the index patients (IPs) were evaluated by a standardized questionnaire for recording bleeding manifestations and by performing conventional coagulation tests. The diagnosis of VWD type 3 was confirmed by VWF antigens less than 5 IU/dL. Direct sequencing of VWF gene (VWF) was carried out to identify causative gene variations. We evaluated the potential consequence of novel splice site and missense variations by predictive computational programs and in silico structural analysis. Results VWF mutations were detected in 46 out of 48 IPs (95.8%), predominantly as homozygous variants. In total, twenty‐nine different gene defects were characterized in this cohort from which 10 (34.5%) are novel. The majority of the mutations were null alleles (66%; including gene conversions, nonsense, splice site variations, small deletions and insertions), and 34% of them were missense substitutions. Conclusion Herein, we reported for the first time, the pattern of gene defects in Pakistani type 3 VWD cohort. We identified a wide heterogeneous mutation spectrum along with variability in the type of bleeding episodes.

Objective: To compare the platelet count, platelet concentration/yield, residual Red blood cells (RBCs) and White blood cells (WBCs) counts in platelet-rich plasma (PRP) samples prepared from the single- and the double-centrifugation protocols. Methods: It was a Cross-Sectional study, conducted at the Department of Hematology & Transfusion Medicine, The Children’s Hospital and UCHS, Lahore from October 2021 to January 2022 including 50 voluntary, healthy individuals of age 20-45 years of both genders, after taking informed consent. Complete blood count analysis of all participants was done initially by drawing 3ml blood in EDTA vial. From all the participants, 20 ml venous blood sample was taken in syringes containing tri-sodium citrate and then shifted to harvest tubes. Group-I comprised of PRP samples prepared by single- centrifugation method. While Group-II samples were prepared by Double-centrifugation method consisting of soft and hard spin. The platelet, RBC and WBC counts in prepared PRP samples were determined by using automated SYSMEX XP-100 hematology analyzer. Platelet yield or Platelet concentration (%) was calculated for samples using formula. The data analysis was done using SPSS version 23. Results: The mean PRP platelet count in Group-I was 594.6±157.4×103/µl whereas in Group-II was 923.06 ± 127.58×103/µl. In Group-I, the mean platelet concentration/yield in PRP was 175.75 ± 55.08% while in Group-II, it was 276.78 ± 112.7%. Significant difference was observed between the platelet counts and platelet concentration/yields from the PRP samples of two Group-s (p < 0.01). Significant difference between the WBCs count was also observed (p < 0.01) with higher WBCs in Group-I PRP. Residual RBCs were almost same among two Group-s. Conclusions: The double centrifugation protocol resulted in higher platelet quantity and yield with less contamination by red and white blood cells than did the single centrifugation protocol for PRP preparation. So, double centrifugation method is beneficial in preparation of autologous as well as allogenic PRP. doi: https://doi.org/10.12669/pjms.39.3.7264 How to cite this: Saqlain N, Mazher N, Fateen T, Siddique A. Comparison of single and double centrifugation methods for preparation of Platelet-Rich Plasma (PRP). Pak J Med Sci. 2023;39(3):634-637. doi: https://doi.org/10.12669/pjms.39.3.7264 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT:The participation of women in blood donation process has been scarce equally in low & high resourced countries. Every year 3.5 million blood donations are collected in Pakistan with very little percentage of females donating blood. Objective: The objective of the study was to assess the knowledge, attitude and beliefs of female attendants accompanying the patients. Study Design: Cross-sectional study. Setting: The Children's Hospital & Institute of Child Health, Lahore, regarding blood donation. Period: 1 st June to 31 st December, 2015. Material & Methods: 306 females of age 16-50 years accompanying a patient in the hospital were randomly selected. After taking informed consent, a self-designed questionnaire validated by Kappa Statistics program was distributed to the participants. Data was analyzed by using SPSS 22. Results: The mean age of female attendants was 29.57 years. 31.7% had never received any education. Only 12.7% had donated blood before. 93.8% had the idea that a blood transfusion is a lifesaving procedure but only half of them knew the common blood groups. 87.3% knew that both males and females can donate blood but 75.5% thought that males are better donors than females. There was a lack of knowledge about certain important facts including transfusion transmissible infections, donation frequency, duration and volume of blood collected during a single session. Majority females were of the view that they are too fragile to donate blood and severe and prolonged anemia can develop after donating blood. Conclusion: There is a need to develop effective interventions for the education & motivation of potential female donors towards blood donation in order to increase the donor turnover rate. Key words:Female, Blood Donation, Participation, Pakistan. Article Citation: Saqlain N, Ahmed N, Ahmed A, Hareem S. Blood donation; Knowledge, attitude and beliefs of the female attendants.

Objective: To determine the frequency of ABO discrepancies in pediatric patients of lymphoma and solid organ tumors and to categorize these discrepancies and their resolution. Study Design: Cross-sectional study. Setting: Department of Hematology & Transfusion Medicine, The University of Child Health Sciences & The Children’s Hospital, Lahore. Period: November 2020 to September 2021. Material & Methods: ABO blood group discrepancies were assessed by tube method of blood grouping, using antisera A, B, AB & D for forward grouping and A, B, and O cells for reverse grouping. Auto control was also run. The resolution techniques were used accordingly. The collected data was checked for its completeness, consistency and accuracy before analysis which was done on SPSS version 26. Results: In this study, a total of 105 subjects were included with mean age of 5.64 ±2.1 years. Among them 72(68.6%) were male and 33(31.4%) were female. Out of total samples processed, three (2.9%) discrepancies found, 1 in female and 2 in male patients. There were two cases of Group I ABO discrepancies, one of which was resolved by elution, antibody screening and identification and other by incubation at 37oC and correlation with previous transfusion history. One case of Group II ABO discrepancy which was resolved by incubation at 40C for 30 minutes. Conclusion: This shows that ABO discrepancies occur in pediatric patients of lymphoma and solid organ tumors. So, the interpretation of forward and reverse ABO blood grouping, identification and resolution of ABO discrepancies in these patients should be done very carefully to avoid any transfusion related adverse reactions.

Aim: To determine frequency of alloimmunization and autoimmunization among the patient of thalassemia with history of multiple blood transfusions. Methods: This study was conducted by consecutive sampling in six months duration from September 2020 to August 2021 at the department of Hematology and blood Transfusion Medicine, of University of Child Health Sciences, The Children's Hospital Lahore. Any patient with a positive Direct antiglobulin test (DAT) was labeled as auto-immunized and a positive indirect antiglobulin test (IAT) was labeled as alloimmunized. Results: Total 90 cases were enrolled with 62% male and 38% females with a mean age 6.04 + 3.3 years (range of age: 7 months to 14 years). Antibody screening and auto-antibodies were positive in 4.4% (n= 4) cases each, and allo-antibodies in 6.7% (n=6) cases (anti-E in 2, anti-C in 2, anti-K and anti-e in 1 each). Among all these patients of beta thalassemia, spleen was enlarged in 71% cases; around half of these were <5 years old. Around 96% had first transfusion <2 years. The frequency of patients positive on DAT, IAT and on anti-body screening was significantly related to the frequency of blood transfusion Practical Implications: Autoantibodies produce a positive direct antiglobulin test (DAT), resulting in hyperhemolysis of red cells in thalassemic patients, which exacerbates the existing alloantibodies factor in children who have received several blood transfusions. The gap between transfusions will be shortened. Routinely, greater emphasis is placed on the identification of allo-antibodies, but auto-antibodies should also be evaluated so that this phenomena may be better understood and handled. This study was conducted to examine the link between a greater transfusion demand and the existence of auto or allo antibodies in Thalassemia patients. I Conclusion: We found Alloimmunization and autoimmunization together not very uncommon in transfusion dependent patients of Thalassemia. The frequency of transfusions is affected in co-existing states. Extended matching, as well as early diagnosis and control of Auto and allo-antibodies both can improve the efficacy of blood transfusion. Keywords: Autoantibodies, IAT, DAT, Coomb’s test, Thalassemia, Allo-immunization

ORIGINAL PROF-3471 ABSTRACT… Background: In 1939 Rh antigen was discovered by Levine and Stetson. Rh system antigens are very immunogenic, they can produce significant Hemolytic Disease of the fetus and Newborn as well as hemolytic transfusion reactions. There are numerous variants of D, the most common subtypes are Weak D and Partial D, now called as abnormal D antigens. The incidence of Rh negativity worldwide varies between 3%-25% and that of weak D antigen ranges from 0.2%-1%. Objectives: To find out the frequency of Rh negativity and weak D antigen among the donors coming to the blood bank of The Children's Hospital & Institute of Child Health, Lahore and to review the clinical significance of weak D antigen in transfusion perspective especially its role in alloimmunization caused by Weak D antigen when transfused to Rh negative individuals. Study Design: Cross-sectional study. Setting: The Children's Hospital and Institute of Child Health, Lahore. Period: 1 st Jan 2015 to 31st May, 2015. Materials and Methods: 6320 healthy donors were randomly selected. All samples were grouped for ABO and Rh-D factor by immediate spin tube technique. All samples found Rh negative, were further processed for weak D antigen with monoclonal anti D sera by using indirect Coomb's technique. The presence of macroscopic or microscopic agglutination was recorded as Rh positive. In case there was no agglutination the mixture was washed 4 times with normal saline. After the last wash, saline was decanted and 2 drops of monoclonal, polyvalent anti human globulin was added. Macroscopic and microscopic agglutination was looked for and any agglutination at this stage was recorded as weak D antigen. Positive control (check cells i.e. washed O positive cells with diluted anti D) and negative control (washed O positive cells) were always put. Results: Among the 6320 healthy donors, 1224(19.4%) were Rh-D negative and 5096(80.6%) were Rh-D positive. Of the 1224 Rh D negative samples, 3 (0.2%) samples found positive for weak D antigen. Conclusion: The frequency of Rh negative blood group was 0.2% among the healthy donors at The Children's Hospital and ICH, Lahore. Although the frequency is low but it's proven by literature that weak D antigen can produce alloimmunization if transfused to Rh-D negative subjects. At the same time the cases of hemolytic reactions reported previously with Weak D antigen have been scarce.