A review on risk assessment of formulation development and technology transfer of COVID-19 vaccines / Una revisión sobre la evaluación de riesgos del desarrollo de formulaciones y la transferencia de tecnología de las vacunas COVID-19

Background: COVID-19 pandemic situation made the pharmaceutical companies develop the vaccine with different formulations in a short period. Objectives: The main objective of the review is to focus on different types of vaccine formulations available globally and the importance of technology transfer in vaccine development associated with potential risks. Results: Research on vaccine development led to various types of vaccines, such as Inactivated vaccines, Live Attenuated vaccines, Ribonucleic acid (RNA) and Deoxyribonucleic acid (DNA) vaccines, viral vector vaccines, and Protein Subunit Vaccines for COVID-19. But the process of vaccine development and technology transfer is lined with various risks and challenges. Through risk assessment, we found some major potential risks involved in product development; this leads to a smoother and more efficient method to develop safe vaccines available for public health. Conclusions: This review will explain the significance of technology collaboration for the faster development of various formulations of vaccines globally

Antecedentes: La situación de pandemia de COVID-19 hizo que las empresas farmacéuticas desarrollaran la vacuna con diferentes formulaciones en un corto período. Objetivos: El objetivo principal de la revisión es centrarse en los diferentes tipos de formulaciones de vacunas disponibles a nivel mundial y la importancia de la transferencia de tecnología en el desarrollo de vacunas asociado con los riesgos potenciales. Resultados: La investigación sobre el desarrollo de vacunas condujo al desarrollo de varios tipos de vacunas, como vacunas inactivadas, vacunas vivas atenuadas, vacunas de ácido ribonucleico (ARN) y ácido desoxirribonucleico (ADN), vacunas de vectores virales y vacunas de subunidades de proteínas para COVID-19. Pero el proceso de desarrollo de vacunas y transferencia de tecnología está lleno de varios riesgos y desafíos. A través de la evaluación de riesgos, encontramos algunos riesgos potenciales importantes involucrados en el desarrollo de productos, lo que conduce a un método más fluido y eficiente para desarrollar vacunas seguras disponibles para la salud pública. Conclusiones: Esta revisión dará una idea de la importancia de la colaboración tecnológica para el desarrollo más rápido de varias formulaciones de vacunas a nivel mundial

Delta-beta thalassaemia in a pathan family

Delta-beta-thalassaemia [macron beta-thalassaemia] is a rare type of thalassaemia which mostly results from deletion of macron and beta genes with preservation of gamma genes. Macron beta-thalassaemia is classified into [macron beta]+ and [macron beta] 0 types. The [macron beta] 0-thalassemia is further divided into G gamma A gamma [macron beta] 0-thalassaemia and G gamma [A gamma macron beta] 0-thalassaemia. In heterozygous state, [macronbeta] 0 mutations give rise to phenotype resembling beta-thalassaemia trait but with raised Hb-F, ranging from 5 to 20%, without a rise in Hb-A2. In homozygotes, the clinical picture is usually that of thalassaemia intermedia and the patients have 100% Hb-F. Workup of a 1-year child suffering from pallor, chronic ill health, and splenomegaly referred to our laboratory with the suspicion of beta-thalassaemia, ultimately resulted in a diagnosis on polymerase chain reaction as having homozygous inversion/deletion G gamma [A gamma macron beta] 0-thalassaemia. Her family members were also investigated

Effect of iron deficiency on the phenotype of beta-thalassaemia trait

The objective of this study was to determine the effect of iron deficiency on Hb-A2 level in beta-thalassaemia trait and to determine the frequency of individuals with beta-thalassaemia trait who could be missed due to concomitant iron deficiency. A total of 120 patients were studied, out of which 23 were iron deficient [serum ferritin < 20 ng/ml]. Mean Hb-A2 in the iron deficient individuals was 4.1 +/- 0.47% as compared to 5.1 +/- 0.58% in the remaining 97 individuals without iron deficiency [p < 0.001]. In the 120 individuals with beta-thalassaemia trait, mean Hb-A2 was 5.8% with range 3 - 6.8% and confidence interval was 95%. In 2 individuals with beta-thalassaemia trait, Iron deficiency was observed and showed Hb-A2 less than 3.5%. These could have been missed while screening by Hb-A2 estimation alone. Co-existence of Iron deficiency and beta-thalassaemia trait may mask the diagnosis of beta thalassaemia trait and such individuals can be missed during screening by Hb-A2 estimation alone

Early diagnosis of dengue virus infection in clinically suspected cases

Objective: Comparison of real time reverse transcriptase polymerase chain reaction [RTPCR] and immunoglobulin M [IgM] capture enzyme linked immunosorbent assay [ELISA] for diagnosis of dengue virus infection in first week of illness in clinically suspected patients of dengue fever

Study Design: Cross sectional study

Place and Duration of Study: Department of haematology, Armed Forces Institute of Pathology [AFIP] Rawalpindi from Jan 2013 to Nov 2013

Material and Methods: A cross sectional study including 68 clinically suspected patients of dengue fever according to the World Health Organization [WHO] criteria. IgM capture ELISA and RT PCR for dengue virus ribonucleic acid [RNA] was performed on samples collected from patients having fever for 1 to 7 days. These were divided into two groups. Patients in group 1 presented with fever of 4 days or less, patients in group 2 had fever of 5 to 7 days duration

Results: In group 1, 72% of the patients were positive by RT PCR while 31% were positive by IgM capture ELISA. In group 2, 43% of the patients were positive by RT PCR while 97% were positive by ELISA

Conclusion: RT PCR can be used for early detection of dengue virus infection in the first few days of fever while IgM ELISA is diagnostic afterwards

Frequency of Gc-globin promoter 158 [C>T] XmnI polymorphism in patients with homozygous/compound heterozygous beta thalassaemia

BACKGROUND: Response to hydroxyurea therapy in homozygous or compound heterozygous beta thalassaemia [BT] has been reported as more favourable in the presence of XmnI polymorphism. The prevalence of XmnI polymorphism may vary with BT phenotypes and genotypes, and differs geographically in distribution. Prevalence of XmnI polymorphism is not known in northern Pakistan. Objective: To determine the frequency of Gc-globin promoter 158 [C>T] XmnI polymorphism [XmnI polymorphism] in patients with homozygous or compound heterozygous beta thalassaemia. MATERIALS: Polymerase chain reaction [PCR] for common beta thalassaemia mutations and Gc-globin promoter 158 [C>T] XmnI polymorphism was performed on 107 blood samples of transfusion dependent beta thalassaemia [BT] patients in Pakistan. One hundred samples of unrelated BT traits and 94 samples of healthy subjects as controls were also analysed for BT mutations and XmnI polymorphism. Results: Out of 301 DNA samples, XmnI polymorphism was detected in 71[24%]; in normal controls, XmnI polymorphism was detected in 34/94 [36%] subjects; while in homozygous/compound heterozygous BT, it was detected in 14/107[13%] patients [Fisher's exact test, p =. 0002]. In heterozygous BT group, XmnI polymorphism was detected in 23/100 subjects [Fisher's exact test, p =. 03 with normal controls, and p =. 049 with homozygous/compound heterozygous BT]. The most common BT genotype was Frame Shift [Fr] 8–9/Fr 8–9, and none of the patients with this genotype had XmnI polymorphism. The second most common genotype was IVSI-5/IVSI-5; 4/26 [15%]. Cases with this genotype had XmnI polymorphism. Conclusion: XmnI polymorphism in homozygous/compound heterozygous BT group is 13%. The most common genotype associated with XmnI polymorphism was IVSI-5/IVSI-5

Frequency of pyruvate kinase [PK] deficiency in neonates with haemolytic anaemia

Objective: To determine the frequency of pyruvate kinase deficiency in neonates presenting with haemolytic anaemia

Study Design: Cross sectional descriptive study

Place and Duration of Study: Haematology department, Armed Forces Institute of Pathology, Rawalpindi [AFIP] from Jan 2011 to Jan 2012

Material and Methods: Study was done in collaboration with neonatology department of Military Hospital. Informed consent from parents of neonates was obtained. Two hundred and twenty five neonates with haemolytic anaemia based on low haemoglobin [<14g/dl], raised reticulocyte counts [>5%] and indirect hyper bilirubinaemia [as per CDC nomogram for evaluation of hyper bilirubinaemia in term neonates] were selected. Qualitative pyruvate kinase enzyme assay was done using Bio vision PK assay kit. Estimation of enzyme was based on generation of pyruvate by addition of substrate with change in optical density [OD] of sample in presence of enzyme. Dilution of standard as recommended by manufacturer was made and standard graph was plotted. The OD was measured using wave length of 570 nm at two points [start and at 25 min]. Cut off limit of less than 25% activity was considered positive for pyruvate kinase deficiency. Confirmation was done by running in parallel negative and positive controls [provided]

Results: Seven [3.1%Confidence Interval: +/- 3.33] out of 225 patients were found deficient. Among these 4 were male and 3 were female neonates. The age range was between 1 to 3 weeks. The mean age of presentation was 1.89 +/- [0.832] weeks

Conclusion: We conclude that pyruvate kinase deficiency is not uncommon in our setup and all patients with congenital non-spherocytic haemolytic anaemia where cause cannot be established should be screened for pyruvate kinase deficiency

Haematological manifestations and frequency of FAB subtypes in patients of acute meyoloid leukaemia: a single center study

To determine the clinical/haematological manifestations and frequency of different subtypes of Acute Myeloid Leukaemia [AML] according to the French-American-British [FAB] classification. Descriptive study. The study was carried out at haematology department of Armed Forces Institute of Pathology [AFIP], Rawalpindi from January 2011 to September 2012. Retrospective review of documents of patient diagnosed to have acute myeloid leukaemia on bone marrow aspiration was done. Patient's age, gender, major signs and symptoms at time of presentation and haematological parameters of peripheral blood and bone marrow were noted. The subtype of AML according to FAB classification was also documented. Data was entered and analyzed in SPSS 16.0. During the selected study duration acute myeloid leukaemia was diagnosed in 173 patients on bone marrow examination. Out of these 123 [71.1%] were males and 50 [28.9%] were females. Thirty [17.3%] of the patients fell in paediatric age group [< 15 years] while the remaining 143 [82.7%] were in adult age category [> 15 years]. The mean age of presentation was 9 years among paediatric patients and 44.5 years among adults. The overall mean age of both these two groups was 38.4 years [3-84 years]. Fever [71.6%], generalized weakness [34.1%] and pallor [23.7%] were the three main complaints of the patients, followed by splenomegaly and lymphadenopathy. The mean total leukocyte count, haemoglobin and platelet count were 57.4 x 10[9]/L, 7.9 g/dL and 54 x 10[9]/L respectively. AML-M[2] was found to be the most frequent FAB AML subtype among 72 [41.6%] paediatric and adult patients. The main signs and symptoms of the patients of AML presenting to our centre were fever, generalized weakness and pallor. AML-M[2] was found to be the most common FAB subtype among AML in children and adults

Calretinin expression in the differential diagnosis of ameloblastoma and keratocystic odontogenic tumour

To determine calretinin expression by immunohistochernistry in ameloblastoma and keratocystic odontogenic tumors [KCOT] and to document the use of calretinin as a differentiating marker between the two lesions. A cross sectional study conducted on previously diagnosed cases of ameloblastoma and Keratocystic odontogenic tumour. Armed forces Institute of Pathology, Rawalpindi Pakistan and duration was one year. [Sep 2009 - Aug 2010]. Twenty cases each of Ameloblastoma and KCOT were retrieved from the record files along with their paraffin embedded blocks. Histological features of all the cases were reviewed on freshly prepared slides and a fresh diagnosis made regardless of the previous diagnosis. The immunohistochemical marker, Calretinin, was applied on both types of cases using the avidin-biotinylated peroxidase complex method.The results were interpreted. In the cases of Ameloblastoma the epithelial tumour nests showed positivity for Calretinin expression. In 85% cases; intense and diffuse staining was observed in more than 80% of the stellate reticulum like cells while 15% cases showed focal and moderate staining patterns. On the other hand KCOT showed contrary results as none of epithelial lining expressed positive staining for Calretinin, [p<0.001]. Calretinin can be used as a useful marker for Ameloblastoma and can be used to differentiate KCOT from Ameloblastoma

Real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

To compare the sensitivity and specificity of Real Time Polymerase Chain Reaction [RT-PCR] with conventional cytogenetics in diagnosis of chronic myeloid leukemia. A cross-sectional, analytical study. The Armed Forces Institute of Pathology [AFIP], Rawalpindi, from December 2010 to January 2012. A total number of 40 patients were studied, in which all were diagnosed as CML on peripheral blood and bone marrow aspiration. The subjects were tested for the presence of Philadelphia [Ph] chromosome by cytogenetics and BCR-ABL fusion gene by RT-PCR. 2-3 ml of venous blood was collected, half in sodium heparin [anti-coagulant] for cytogenetics and half in EDTA for PCR. For cytogenetics, cells were cultured for 72 hours in RPMI 1640 medium and examined by arresting in metaphase using Colchicine to identify Philadelphia chromosome. For PCR, RNA extraction was done by Tri Reagent LS [MRC, USA] and cDNA was synthesized using reverse transcriptase and gene specific primer. RT- PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of cytogenetics and RT PCR were compared. Out of the 40 patients, PCR showed 37 [92.5%] were positive and 3 [7.5%] were negative for BCR-ABL fusion gene, whereas in cytogenetics 28 [70%] were positive for Ph chromosome and 12 [30%] were negative for Ph chromosome. Sensitivity and specificity of cytogenetics was 75.6% and 100% respectively. Real time PCR as compared to cytogenetics is less tedious, gives quick results, does not require multiple sampling due to culture failure and can be done on peripheral blood

Acute Lymphoblastic Leukaemia: Clinicohaematological Features, Laboratory Characteristics and Prognostic Factors: a Single Center Experience

To study clinico-haematological features, Laboratory results and prognostic factors in patients of acute lymphoblastic leukaemia. Descriptive study. This study included all newly diagnosed cases of acute lymphoblastic Leukaemia coming to Armed Forces Institute of Pathology Rawalpindi from Jun 2008-Feb2010. The detailed clinical history with physical findings were charted on the proforma. About 3ml blood from each patient was taken in EDTA container. The blood was analyzed on Haematology analyzer Sysmex KX 21. Quality control was maintained by running normal and abnormal controls. Bone marrow aspiration was done at the time of diagnosis. Five push smears were made from each case; 2 for leishman stain, one for Sudan black B, one for periodic cid schiff, and one for acid phosphatase. The common clinical features in children were pallor [100%], fever [93%], hepatomegaly [70%], splenomegaly [64%], lymphadenopathy [58%], bleeding manifestations [27%] and bone pain [9%]. Pallor [100%] and fever [89%] were also common manifestations in adults. Initial high white cell count [> 50x109/l] was observed in 9 [12%] patients. Three patients showed hyperleucocytosis [> 100x109/l]. Haemoglobin < 8gm/dl was seen in 30 11%] patients and platelet count less than 20x109/l was observed in 8 [10.8%] cases. About 9 [12%] patients showed pancytopenia. According to French-American-British [FAB] criteria ALL-L1 was the commonest FAB type [81%], followed by L2 [16%] and L3 [3%] in children while ALL L2 was high among adult age group. We found that ALL is a frequent childhood hematological malignancy in our setting and is more prevalent in males both in children and adults. ALL- L I type being more common than other types of ALL. Considering the prognostic factors of age, WBC count, lymphadenopathy, T immunophenotyping an FAB classification; most of our patients constitute a better prognostic group

Alterations of P53 [gene mutation and protein expression] in oral epithelial dysplasia

Oral Squamous Cell Carcinoma [OSCC] is usually preceded by precancerous lesions. These lesions appear white or red clinically [Leukoplakia and Erythroplakia respectively] and show dysplastic epithelial changes on histopathological examination [Abbas et al, 2007]. If p53 alterations [gene mutations and protein expression] in premalignant lesions are detected and treated in their early stages, it might help in prevention of progression to cancer [Patton et al, 2008]. Aim was to determine the frequency of p53 gene mutation and protein expression in oral epithelial dysplastic lesions. This was a descriptive study carried out at the Armed Forces Institute of Pathology [AFIP], Rawalpindi and was of one year duration from 8[th] May 2010- 5[th] May 2011. Thirty cases of oral epithelial dysplasia [OED] were retrieved from the record files. Some fresh /frozen sections were also included. Gene p53 mutation was detected in these cases by PCR-SSCP Analysis. DNA was extracted from the formalin-fixed and paraffin-embedded tissue sections and fresh /frozen sections. DNA thus extracted was amplified by polymerase chain reaction. The amplified products were denatured and finally analyzed by gel electrophoresis. Gene mutation was detected as electrophoretic mobility shift. The immunohistochemical marker p53 was applied to the same 30 cases andproteinp53 expression was recorded. Mutations of the p53 gene were detected in 20% of the dysplastic lesions. Immunohistochemical staining ofmarkerp53 was positive in 60% of the cases. Gene p53 mutation and protein expression was not coexistent

Comparison of real time polymerase chain reaction with microscopy and antigen detection assay for the diagnosis of malaria

To determine the sensitivity of a real time polymerase chain reaction [PCR] for malaria diagnosis and to compare its accuracy with microscopy and an antigen based rapid diagnostic test [OptiMal]. Cross-sectional analytical study. Military Hospital, Armed Forces Institute of Transfusion and Armed Forces Institute of Pathology, Rawalpindi, from July to December 2011. Venous blood samples of 300 clinically suspected patients of malaria were tested for malaria parasite by microscopy and OptiMal; and malaria parasite index was calculated for the positive samples. Plasmodium genus specific real time PCR was performed on all specimens, targeting small subunit rRNA gene. Diagnostic accuracy of three tests was compared and cost analysis was done. Out of 300 patients, malaria parasite was detected in 110, 106 and 123 patients by microscopy, OptiMAL and PCR respectively. Real time PCR was 100% sensitive while microscopy and OptiMal had sensitivity of 89.4% and 86.2% respectively. All methods were 100% specific. The cost per test was calculated to be 0.2, 2.75 and 3.30 US$ by microscopy, OptiMal and PCR respectively, excluding the once capital cost on PCR equipment. Genus specific real time PCR for the diagnosis of malaria was successfully established as a highly sensitive and affordable technology that should be incorporated in the diagnostic algorithm in this country.

Immunoglobulin gene rearrangement in detection of minimal residual disease in acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia [ALL] is mainly a childhood malignancy but affects both children and adults. The study was conducted to evaluate a qualitative PCR based method for detection of clonal immunoglobulin gene rearrangement as a marker of minimal residual disease in patients of acute lymphoblastic leukaemia at the end of induction. It was a descriptive study conducted at Armed Forces Institute of Pathology, Rawalpindi from Aug 2009 to Feb 2010. For prospective analysis, genomic DNA was extracted from peripheral blood/bone marrow aspirates and unstained bone marrow smears. A total of 50 patients of acute lymphoblastic leukaemia who showed positive immunoglobulin gene rearrangement by qualitative PCR at the time of diagnosis were included. These patients were then investigated for minimal residual disease at the end of induction. PCR amplification of the IgH gene was done by a VH primer homologous with a highly conserved sequence near the 3´ end of the FR3 region and a consensus sequence JH primer. Test for minimal residual disease was conducted by PCR amplification of DNA from remission marrow cells [at day 29 of chemotherapy] with the help of the primer sets used at the time of diagnosis. The amplified DNA was seen by electrophoresis on 6% polyacrylamide gel. A sharp clonal band ranging from 90-200 bp indicated a positive reaction. Of 50 patients, 28 [56%] were positive for Ig gene rearrangement on PCR at the end of induction, 17 [34%] patients were found to be negative for minimal residual disease, 2 [4%] patients died during induction therapy, and 3 [6%] patients did not come for follow-up. Molecular approaches have allowed us to detect low level of residual disease which is not detected by cytomorphological methods. Minimal residual disease [MRD] by PCR used in this study would definitely help in monitoring of MRD in all patients with leukaemia

Role of genetic counselling in prenatal diagnosis of p-thalassaemia in Pakistan

To compare the response towards prenatal diagnosis [PND] of [3-thalassaemia, in individuals who had not received genetic counselling and a genetically counselled population. Cross-sectional survey. Department of Haematology, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from March 2009 to December 2010. Using non-probability consecutive sampling, a total of 176 individuals having thalassaemic children, were interviewed regarding PND of thalassaemia, by using a structured questionnaire. Forty two individuals were taken as controls as they had received genetic counselling for PND, whereas the remaining 134 were taken as cases. Responses towards PND were compared using chi-square test. Odds ratio was also calculated for subsequent PND utilization. Seventy [52.2%] cases and 42 [100%] controls were aware of the availability of PND in Pakistan. This difference in awareness was statistically significant [p < 0.001]. In the controls, 40 [95.3%] individuals were aware of the appropriate timing of the test, in contrast to 52 [39%] cases [p < 0.001]. PND was used in subsequent pregnancies by 50 [37.3%] cases and 32 [80%] controls [p < 0.001]. The calculated odds ratio for subsequent PND utilization was 5.37. The study reflects a very positive attitude of genetically counselled thalassaemia affected families towards PND. For better utilization of PND, genetic counselling services should be available at all health strata

Frequency of Janus associated kinase 2 [JAK2] mutation in patients of BCR-ABL negative myeloproliferative neoplasms

To determine the frequency of Janus associated kinase 2 mutation in the patients of BCR-ABL negative classical myeloproliferative neoplasms. Cross-sectional descriptive study. Molecular Department of Haematology, Armed Forces Institute of Pathology [AFIP], Rawalpindi from Jul 2011 to Jul 2012. Ninety three consecutive patients of Polycythaemia vera [PV], Essential thrombocythaemia [ET] and Idiopathic myelofibrosis [IMF] diagnosed by the conventional haematological criteria were included in the study. All patients were screened for G-T point mutation [V617F] in the JAK2 gene on chromosome 9 by an allele specific PCR. Out of the 93 myeloproliferative neoplasm [MPN] patients, 33[35%] had polycythaemia vera, 36[39%] had essential thrombocythaemia and 24[26%] had idiopathic myelofibrosis. JAK2 mutation was seen in 64/93 [69%] patients including 33/33[100%] in PV, 19/36[52.6%] in ET and 12/24[50%] in IMF. Classical myeloproliferative neoplasms are an important group of heamatological disorder in our country. JAK2 gene mutation is seen in significant proportion of these disorders [69%]. JAK2 mutation analysis can be used to differentiate between polycythemia vera and secondary polycythemia in most cases with near certainty, where it was found in 100% of the cases

Silent mutations of thalassaemia - a diagnostic dilemma

A case of thalassaemia intermedia resulting from compound heterozygosity between Fr8-9 and Cap+1 mutation is presented. Patient's father had undergone premarital thalassaemia screening and was declared free of thalassaemia due to normal HbA[2] levels. We aim to discuss the clinico-haemtological features and diagnostic approach for Cap+1 mutation in carrier as well as compound heterozygous state with a beta[0] mutation

Feasibility and safety of transabdominal chorionic villus sampling

Chorionic Villus Sampling [CVS] is the technique of choice for prenatal diagnosis prior to 12 weeks gestation. The objective of this study was to determine the feasibility, and pattern of complications following first trimester Trans-abdominal Chorionic Villus Sampling [TA-CVS]. This was a descriptive study conducted in the Obstetrics and Gynaecology Department Military Hospital [MH] Rawalpindi from Jan 2007 to July 2008. Couples at risk of giving birth to a child with genetic disorder were identified and counselled. Trans-abdominal Chorionic Villus Sampling was done using double needle technique under ultrasound guidance. Immediate and late complications were followed up. Data was analysed using SPPS-10. On 200 cases chorionic villus sampling was done as an outdoor procedure. Most common indication was thalassaemia trait 75 [37.5%]. Most procedures were done between 12-13 weeks. All placental positions including 104 [52%] posterior and 71 [35.5%] anterior were approachable. Most aspirations were easy, however, in 30 [15%] the aspiration was difficult. Overall success rate was 100%. In 158 [79%] of the cases sample yield was good. One [0.5%] patient had vaginal bleeding and three [1.5%] had placental haematoma formation. Most patients [84%] experienced mild pain during the procedure. The procedure related miscarriage occurred in 2 [1%] patients while another patient developed this complication after 6 weeks. First trimester TA-CVS is an accurate and safe invasive prenatal diagnostic procedure. Placentas in almost any position can be approached without any significant risk to mother and the foetus.

Mixed donor chimerism in non-malignant haematological diseases after allogeneic bone marrow transplantation

To determine the frequency of mixed donor chimerism in patients of non-malignant haematological diseases after allogeneic bone marrow transplant. A cross-sectional, observational study. Department of Haematology, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from July 2010 to June 2011. Methodology: Donor chimerism was assessed in patients of aplastic anaemia and beta-thalassaemia major who underwent allogeneic bone marrow transplantation [BMT]. Peripheral blood samples were used to assess chimerism status by analysis of short tandem repeats [STR]. In patients where pre-transplant blood sample was not available, swab of buccal mucosa was used for pre-transplant STR profile. A standard set of primers for STR markers were used and the amplified DNA was resolved by gel electrophoresis and stained with silver nitrate. The percentage of donor origin DNA was estimated by densitometer. Out of 84 patients, 52 [62%] were males, while 32 [38%] were females. In patients of beta-thalassaemia major, 31 [62%] developed mixed donor chimerism [MC], 13 [26%] developed complete donor chimerism [CC] and 6 [12%] had graft failure. In aplastic anaemia, 17 patients [50%] achieved MC, 13 [38.2%] had CC and 4 [11.8%] developed graft failure. The combined frequency of mixed donor chimerism for both the diseases was 58.3%. D3S1358 was the most informative STR marker in these patients. Majority of the studied patients developed mixed donor chimerism following bone marrow transplantation, whereas only a minor percentage of the patients had graft failure. Analysis of D3S1358 was the most informative in assessing donor chimerism in patients who underwent BMT

Frequency of janus associated kinase 2 [V617F] mutation in patients of polycythemia vera

To determine the frequency of Janus associated kinase 2 [JAK2] mutation in patients of polycythemia vera [PV]. Descriptive cross-sectional. Haematology Department, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from January 2008 to December 2009. Forty-six consecutive patients of PV diagnosed by the conventional haematological criteria were included in the study. Blood samples of all patients were screened for G-T point mutation [V617F] in the JAK2 gene on chromosome 9 by an allele specific polymerase chain reaction [PCR]. JAK2 V617F mutation was found in 43 out of 46 patients [93.5%] with PV. Among them, 30 were males [65.2%] and 16 were females [34.8%]. Mean TLC in patients with PV was 16.5 +/- 9.1 x 109/L, mean haemoglobin [Hb] was 17.8 +/- 2.0 g/dl, mean platelet count was 531 +/- 261 x 109/L, mean PCV was 57.9 +/- 6.3 l/l, mean MCV was 78.8 +/- 11.0 fl and mean MCH was 24.4 +/- 4.8 pg. Peripheral blood mutation screening for JAK2 V617F can be incorporated into the initial work up of patients suspected to have polycythemia as this mutation is present in majority of such patients

Real time polymerase chain reaction for the detection of malarial parasite

To evaluate the sensitivity and specificity of real time polymerase chain reaction [PCR] for the detection of Malarial parasite. Descriptive cross-sectional study. The Armed Forces Institute of Pathology [AFIP], Rawalpindi, from April to June 2010. A total of 60 Leishman stained blood films with clinical suspicion of malaria were studied by light microscopy for detection of malaria parasite [MP]. The samples were also subjected to real time PCR for the small subunit [SSU] rRNA gene of MP found in all the four subspecies of Plasmodium. Real time PCR was done by the Taqman probe method. One sample positive for MP was serially diluted with ABO compatible blood, and light microscopy and real time PCR were performed on all dilutions. Results of light microscopy and real time PCR were compared. Sensitivity and specificity were calculated using PCR as the gold standard. PCR detected MP in 33 samples with sensitivity and specificity of 100% whereas light microscopy could detect MP in 30 samples. Sensitivity and specificity of light microscopy was 90.9% and 100% respectively. In the serially diluted blood sample, MP was visible at 1/16 dilution whereas the PCR showed positive results even at 1/512 dilution. Real time PCR is more sensitive than light microscopy for the detection of malarial parasite