Comparative analysis of fluorescence in situ hybridization and real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

Objective: To compare the sensitivity and specificity of fluorescence in situ hybridization [FISH] with real time polymerase chain reaction [RT-PCR] in the diagnosis of Chronic Myeloid Leukemia [CML]

Study Design: A cross-sectional, analytical study

Place and Duration of Study: Haematology Department, Armed Forces Institute of Pathology, Rawalpindi, from January 2012 to February 2014

Methodology: A total number of 87 patients of CML were studied. The diagnosis was made on the basis of clinical history, peripheral blood and bone marrow aspiration. These patients were tested for the presence of BCR-ABL1 fusion gene by RT-PCR and FISH. About 5 ml of venous blood was collected, half was taken in heparin for FISH and half in ethylenediamine tetra-acetic acid [EDTA] for CBC and PCR. For FISH, cells were cultured for 24 hours in RPMI 1640 medium and evaluated using BX51 fluorescence microscope for dual fusion signal of yellow colour. Samples having 20 or more interphases positive for dual fusion signals were taken as positive. 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 RT-PCR and FISH were compared

Results: Out of the 87 patients, 85 [97.7%] were PCR positive and 2 [2.3%] were PCR negative, whereas in FISH 83 [95.4%] were positive and 4 [4.5%] were negative. Sensitivity and specificity of FISH was 97.6% and 100%, respectively

Conclusion: FISH is a reliable supplementary method to PCR for detection of BCR-ABL1 fusion gene in the diagnosis of CML

Clinico-haematological features of paroxysmal nocturnal haemoglobinuria

The aim of this study was to determine the frequency of various clinico-haematological features in patients suffering from paroxysmal nocturnal haemoglobinuria [PNH]. It was an observational study carried out from October 2008 - January 2016. All the patients of PNH, diagnosed on the basis of clinical and laboratory findings and confirmed by CD55 and CD59 deficiency on red cells by means of flow cytometry, were included in the study. A total of 22 patients were diagnosed which included 18 [81.8%] males and 4 [18.1%] females. Median age was 27 years. Pallor, fever, fatigability and haemoglobinuria were the most common clinical features. Pancytopenia was seen in 13 [59.09%] and hypocellular marrow was found in 14 [63.6%] patients. One patient presented with Budd Chiari syndrome

Impact of medical autopsy on final diagnosis

Objective: To determine the difference between diagnosis and misdiagnosis after medical autopsy

Study Design: Prospective study

Place and Duration of Study: Histopathology Dept Army Medical College Rawalpindi and Military Hospital [MH] Rawalpindi from Jan 2009 to May 2012

Material and Methods: A retrospective cross sectional descriptive study of medical autopsies was conducted on patients at Histopathology Department Army Medical College and MH Rawalpindi who expired at or was brought in dead at MH Rawalpindi during a 41 months period between January 2009 to May 2012. Permission from the ethical committee was obtained for the study. Autopsy for medical purpose was performed on the deceased after receiving written consent from the next of kin. Medical autopsies were performed to determine and find the medical cause of death and to evaluate any disease or injury that may be present. Total of 72 medical autopsies were conducted during the above period. All these consecutive autopsies were included in the study. Ratio of total autopsies done was 0.17% of total hospital deaths at MH Rawalpindi in the duration of study. All subjects were male, military persons, aged between 19 and 50 years. Mean age was 35.5 years. Data was analyzed in excel. Descriptive statistics was applied on qualita ive variables. Frequency and parentages was used

Results: Ante mortem diagnosis confirmed as correct on total of 25/72 Autopsies. Clinically missed / wrong diagnosis was found on 47/72 autopsies

Conclusion: Medical autopsy even today in the environment of a tertiary care hospital has irrefutable contribution in establishing final diagnosis and determining errors and omissions. Consequently it has pivotal role in continued improvement in medical care and in study of evolving disease patterns in real time

Diagnostic accuracy of serum iron and total iron binding capacity [TIBC] in iron deficiency state

Objective: To determine the diagnostic accuracy of serum iron and total iron binding capacity [TIBC] in detection of iron deficiency

Study Design: Descriptive, analytical study

Place and Duration of Study: Department of Chemical Pathology and Endocrinology, from January 2013 to October 2015

Methodology: Data of 1,815 patients with results of serum iron, TIBC and ferritin from January 2013 to October 2015 was retrieved from Laboratory information System [LIMS] of AFIP. Diagnostic Accuracy Studies [STARD] guidelines were followed. Subjects of either gender, aged 1 - 68 years were included. Cases with raised serum ferritin levels [male > 336 ng/ml, female > 307 ng/ml] were excluded. Serum Ferritin was taken as gold standard with specificity of 99% and sensitivity of 80% at concentration of 30 ng/ml. Transferrin saturation was determined by dividing serum iron by TIBC and multiplying by 100

Results: Out of 1,815 subjects, 931 [51.29%] were males and 884 [48.71%] were females. The median age of the patients were 29.1 years [Inter-quartile range, IQR 19.1]. Taking ferritin as gold standard, the sensitivity and specificity of serum iron was 63.5% and 38.6%, respectively; while that of TIBC was 64.5 % and 42.8%, respectively. Ferritin showed poor correlation with iron, TIBC and transferrin saturation

Conclusion: Serum iron and TIBC give no additional information in the diagnosis of iron deficiency anemia and these tests are redundant for the diagnosis of iron deficiency state, if serum ferritin is available

Placental morphology at different maternal hemoglobin levels: a histopathological study

To evaluate the histopathological parameters of the placenta like weight, infarct and syncytial knots, at different maternal hemoglobin levels, in both qualitative and quantitative manner. Descriptive study. Army Medical College, National University of Sciences and Technology in collaboration with Department of Obstehics and Gynecology, Military Hospital, Rawalpindi, Pakistan, from December 2011 to November 2012. A total of 75 placentas were included, that were collected from full term mothers at the time of childbirth. Placental weight was taken without umbilical cord and gross placental infarcts were noted. Samples of placental tissue were taken and stained by haematoxylin and eosin [H and E]. Microscopic study was done to evaluate placental infarcts and syncytial knots. Mean placental weight at normal and low maternal hemoglobin was 581.67 +/- 83.97 g and 482.58 +/- 104.74 g respectively. Gross placental infarcts were found in all cases having low maternal hemoglobin concentration [60% cases]. Syncytial knots were found in all placentas but they were considerably more at decreasing levels of maternal hemoglobin [19.79 +/- 5.22]. The present study showed decrease in placental weight, increase in placental infarcts and syncytial knot hyperplasia at low maternal hemoglobin concentration, displaying adaptive alterations

histopathologic analysis of chronic inflammatory infiltrate in patients of H. pylori associated chronic gastritis

To determine the relationship between H. pylori density with severity of chronic inflammatory infiltrate. A cross-sectional study. The study was carried out in the Department of Pathology [Histopathology], Army Medical College, National University of Sciences and Technology [NUST] Islamabad, from Nov 2011 to Nov 2012. Gastric antral biopsies of H. pylori associated chronic gastritis were included in the study. Demographic characteristics and relevant clinical information were collected. First hundred biopsies of H. pylori associated chronic gastritis were assessed for density of H. pylori and chronic inflammatory infiltrate. Histopathological features like lymphoid aggregates, ulcer slough, superficial epithelial damage, dysplasia and nuclear reactive changes were simply assessed in case of their presence or absence. A sigruficant moderate positive correlation was found between grades of H. pylori and chronic inflammatory infiltrate [rs= 0.636]. Insigruficant correlation was found with lymphoid aggregates, superficial epithelial damage, dysplasia and nuclear reactive changes. In conclusion this study corroborated the determination of histopathological parameters and depicted that, the greater the density of H. pylori mfection, the greater the degrees of chronic inflammatory infiltrate

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

Sensitivity Pattern of Second Line Anti-Tuberculosis Drugs against Clinical Isolates of Multidrug Resistant Mycobacterium Tuberculosis

Objective: To determine the current sensitivity pattern of second line anti-tuberculosis drugs against clinical isolates of Multidrug Resistant Mycobacterium tuberculosis [MDR-TB]. Study Design: A cross-sectional study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from November 2011 to April 2013. Methodology: Samples received during the study period were processed on BACTEC MGIT 960 system for Mycobacterium tuberculosis [MTB] culture followed by first line drugs susceptibility testing of culture proven MTB isolates. On the basis of resistance to rifampicin and isoniazid, 100 clinical isolates of MDR-TB were further subjected to susceptibility testing against amikacin [AMK], capreomycin [CAP], ofloxacin [OFL] and ethionamide [ETH] as per st and ard BACTEC MGIT 960 instructions. Results: Out of 100 MDR-TB isolates, 62% were from male patients and 38% from female patients. 97% were sensitive to AMK, 53% to OFL, 87% to CAP; and 87% were sensitive to ETH. Conclusion: The majority of the MDR-TB isolates showed excellent sensitivity against AMK, CAP and ETH. However, sensitivity of MDR-TB isolates against fluoroquinolones like OFL was not encouraging. Key Words: Multidrug resistant tuberculosis. Mycobacteria growth indicator tube. Second line anti-tuberculosis drugs. Amikacin. Capreomycin. Ethionamide

Frequency of different causes of pancytopenia in a tertiary care hospital

To determine the frequency of different causes of pancytopenia on bone marrow examination. Descriptive study. The study was carried out at Haematology [pathology] department of Army Medical College, National University of Sciences and Technology [NUST] and Military Hospital Rawalpindi from Jan 2012- Dec 2012. Total 67 cases of pancytopenia were included in the study. Bone marrow aspiration was done using 16 GLP needle and biopsy was done by using 11 G Trephine biopsy needle. Out of 67 patients, [15%] were children and [52%] were adults. Among children leishmaniasis and hypersplenism were the most common causes [20%] of pancytopenia followed by acute leukemia [3.8%], aplastic anaemia [6.7%] and megaloblastic anaemia [6.7%]. Among adults megaloblastic anaemia was the most common cause [40.4%] followed by lymphoproliferative disorder [15.4%], hypersplenism [7.7%], aplastic anaemia megaloblastic anaemia, acute leukemia and myelodysplasia. Major causes of pancytopenia in children were leishmaniasis and hypersplenism where as in adults they were megaloblastic anaemia and lymphoproliferative disorders

Frequency of different causes of pyrexia of unknown origin on bone marrow examination in a tertiary care hospital

To determine the frequency of underlying causes of pyrexia of unknown origin on bone marrow examination. Descriptive study. The study was carried out at Hematology department [pathology] of Army Medical College, National University of Sciences and Technology [NUST] and Military Hospital Rawalpindi [during the period of one year] from Jan 2012-Dec 2012. Total of 94 patients reporting with pyrexia of unknown origin at MH Rawalpindi underwent bone marrow examination. Bone marrow aspiration procedure was done from posterior superior iliac spine in patients older than one year while tibial tuberosity was used in patients less than one year of age. Lumbar puncture needle of 16 G was used for bone narrow aspiration and trephine biopsy was done by using 11 G trephine biopsy needle. In children, commonest causes observed were acute lymphoblastic leukaemia in 7 [23.3%], marked haemophagocytosis in 4 [13.3%] and visceral leishmaniasis in 4 [13.3%] patients. In adults, commonest causes included megaloblastic anaemia in 13 [20.3%], lymphoproliferative disorders in 8 [12.5%] and hypersplenism in 5 [7.8%] patients. This study concludes that causes of pyrexia of unknown origin vary with age of the patient. The most frequent causes of pyrexia of unknown origin observed in children were acute lymphoblastic leukaemia, marked haemophagocytosis, and visceral leishmaniasis where in adults main causes were megaloblastic anaemia, lymphoproliferative disorders and hypersplenism

High prevalence of protein C, protein S, antithrombin deficiency, and factor V Leiden mutation as a cause of hereditary thrombophilia in patients of venous thromboembolism and cerebrovascular accident

To determine the frequency of Protein C, Protein S [PC and PS], antithrombin deficiency [AT III] and Factor V Leiden mutation [FVL] as a cause of thrombophilia in the patients with venous thromboembolism [VTE] and cerebrovascular accident [CVA]. It was an observational study conducted at Department of Haematology, Armed Forces Institute of Pathology [AFIP], Rawalpindi, Pakistan. All patients referred for thrombophilia screening from July 2009 to June 2012 were screened. Patients with evidence of VTE or CVA were screened for PC and PS, AT III deficiency, and FVL. Total 404 patients of age between 1-71 years mean 33 +/- 14 with male to female ratio of 2.4:1 had evidence of thrombophilia. Two hundred eighteen [54%] patients presented with CVA, 116 [29%] with deep vein thrombosis [DVT], 42 [10.5%] with pulmonary embolism [PE], and 28 [7.5%] with portal or mesenteric vein thrombosis [PV]. Protein C and S deficiency was detected in 35/404 [8.7%], AT III in 9/404 [2%], and FVL in 25/173 patients [14.5%]. The findings were suggestive of a significant association of FVL mutation for developing DVT [OR=11.0, 95% C I 4.6-26.3], CVA [OR=5.7, 95% C I 2.1-15.1], and PV [OR=5.4, 95% C I 1.3-21.9]. PC and PS deficiency was a significant risk factor for developing PE [OR=3, 95% C I 0.8-11.4]. FVL mutation and Protein C and S are the leading causes of thrombophilia with strong association of Factor V Leiden mutation as risk for developing DVT

Thrombotic thrombocytopaenic purpura in a patient presenting with sickle cell -hemoglobin S disease vaso-occlusive crises and acute chest syndrome

The association of sickle cell disease [SCA] vaso-occlusive crises and Thrombotic thrombocytopaenic purpura [TTP] has been rarely described. Here we report a case of sickle cell - haemoglobin S disease [HbSS] in which a patient presented with painful vaso-occlusive crises and acute chest syndrome and TTP which was a diagnostic challenge. We also conducted a Medline search to review reported cases previously

Atypical mayer - rokitansky - kuster - hauser syndrome, hyperprolactinemia, hirsutism and multinodular goiter with obstructive symptoms

Mayer-von Rokitansky-Kuster-Hauser syndrome [MRKH], comprises of combined hypoplasia of the vagina and the uterus, in addition it may be associated with congenital anomalies of the urinary tract and the skeleton. Its main clinical presentation is primary amenorrhoea in the presence of normal secondary sexual characteristics. Multi nodular goiter [MNG] is one of the most prevalent thyroid disorders worldwide and sometimes impairs health and well being. We report a 33-year-old Saudi female with features of the atypical form of Mayer-von Rokitansky-Kuster-Hauser syndrome [MRKH] syndrome, large multinodular goiter with compression of trachea causing obstructive symptoms, hirsutism and hyperprolactinaemia. Although the concurrence of MRKHS and MNG appears to be coincidental, this association has not been previously reported and the association with endocrine abnormalities such as hyperprolactenaemia or hirsutism is rarely described

Outcome of combination antiviral therapy in chronic hepatitis C virus infection during therapy of acute lymphoblastic leukemia

Chronic hepatitis C virus [HCV] infection is not uncommon in patients with acute leukemia due to frequent blood transfusions. The treatment of HCV in patients with acute leukemia can produce profound immune dysfunction with the risk of severe cytopenia. We report the case of a young man who was treated with combined therapy of peginterferon alpha 2a and ribavirin for HCV while he was on maintenance anti-leukemic treatment. The patient required reduction in the dose of peginterferon alpha 2a and the addition of filgrastim due to neutropenia. Therapy for HCV was continued for 72 weeks and at the end of therapy, the patient had undetectable HCV RNA. The patient maintained a sustained viral response two years after the end of therapy and developed complete remission of leukemia, whereupon his anti-leukemic therapy was also discontinued. We recommend conducting further large prospective studies in HCV patients treated for leukemia to determine the safety and efficacy of antiviral therapy in this group of patients

Eosinophilic Gastritis; An unusual and overlooked cause of chronic abdominal pain

Eosinophilic gastritis is an extremely rare disorder. The disease is associated with eosinophilic infiltration of various layers of gastrointestinal tract along with significant peripheral eosinophilia and increased Immunoglobulin E [IgE]. We report a case of 37 year old Saudi male who presented with chronic non-specific upper abdominal pain. On initial workup, the diagnosis was missed. However the diagnosis was established after subsequent work up in Gastroenterology clinic. Our case demonstrates that patients with uncharacteristic abdominal pain who are unresponsive to conventional treatment, rare illnesses like eosinophilic gastritis should be considered. We also aim to review the clinicopathological features, differential diagnosis and various treatment options of this disorder. To the best of our knowledge, this disease has not been previously reported from Saudi Arabia

Frequency of cytogentic abnormalities in patients of Acute Myeloid Leukaemia

Cytogenetic analysis performed at diagnosis is considered to be the most valuable prognostic factor in acute myeloid leukaemia [AML]. Cytogenetic abnormalities which indicate a good prognosis include t[8; 21], inv[16] and t[15;17]. Normal cytogenetics carries average risk in AML. Patients with AML that is characterized by deletions of the long arms or monosomies of chromosome 5 or 7 or by abnormalities of 11q23 have particularly poor prognosis. To determine the frequency and type of cytogenetic abnormalities in Pakistani patients of AML. Design: Descriptive study. Subjects and Thirty six patients of acute myeloid leukaemia were referred to the department of Heamatology, Armed Forces Institute of Pathology, Rawalpindi for cytogenetic studies during the period from March 2001 to September 2004. Five ml of venous blood was collected by venesection in vacutainer containing sodium heparin as anticoagulant. Blood was cultured on RPMI-1640 medium enriched with L-glutamine and foetal bovine serum. Phytohaemagglutin was used as T-cell mitogen. The cultures were incubated for 72 hours at 37°C. Mitoses were arrested in metaphases by colchicine. The cells were harvested and slides were made. Slides were aged and trypsin digestion was done. Slides were stained with Giemsa stain. Twenty metaphases were analysed under the microscope and the observations were recorded. In 10 patients' culture failed to yield evaluable metaphases. Out of 26 evaluated patients, cytogenetic abnormalities carrying good prognosis were seen in 6[23%] patients t[8;21] in 3 cases, t[15;17] in 2 and inv[16] in one patient. Normal karyotype carrying standard risk was seen in 17[65.4%] patients. Whereas abnormalities carrying poor prognosis were seen in only 3[11.6%] patients. These comprised 2 cases of trisomy 8 and one of dup [3][q21; q26]. This study reveals that majority of Pakistani patients with AML belong to good [23%] or standard [65.4%] risk groups. Only 11.6% patients belong to poor risk group