Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by diverse clinical manifestations, including bone marrow failure, intravascular haemolysis and thrombophilia1 2 3
Thrombosis is the primary cause of mortality in PNH, and it accounts for 40-67 per cent of all deaths4 thrombosis 5 6 , 7 thrombosis 6
Thrombosis provides a good model to do a population-based screening for PNH since deep vein thrombosis (DVT) occurs in one per cent of the adult Indian population after the age of 40 yr8 thrombosis is scarce from India and nil from western Rajasthan.
The present study screened for the presence of PNH clones by flow cytometric immunophenotyping in cases of DVT to study the prevalence of PNH in cases of DVT from western Rajasthan.
Material & Methods
The present cross-sectional study was conducted in the departments of General Medicine and Pathology & Laboratory Medicine, All India Institute of Medical Sciences, Jodhpur, a tertiary care centre of western Rajasthan, from August to October 2019. The study protocol was approved by the Institutional Ethics Committee.
A total of 61 adult patients, diagnosed with DVT, but without any provoking factors such as history of trauma, malignancy, recent pregnancy or hormonal therapy were included in the study after obtaining a written informed consent from all the study participants.
Sample collection and processing : Peripheral blood samples (3 ml) were collected and analyzed as per the protocol described earlier9
The blood samples were processed directly if the observed TLC was within the normal range (TLC - 4000-11,000/mm3 ). In case of low TLC, the samples were centrifuged at 200 g for five minutes and excess plasma was removed, maintaining the amount of plasma the same as the red cell pellet. Samples were again mixed properly before processing. Two tubes were used: Tube A (for granulocytes and monocytes): fluorescein-labelled proaerolysin (FLAER)-Alexaflour488/CD45-KromeOrange(B36294)/CD33-APC(IM2471)/CD24-PC5.5(B23133)/CD14-PE (A07764); and Tube B (for RBCs): CD235a-APCAlexaF750(A89314)/CD59-PE(B68140). If a positive PNH clone was found on granulocytes as well as monocytes, processing was done for red blood cells as well.
Flow cytometric analysis : Processed samples were acquired on Navios Flow Cytometer (Beckman Coulter, USA) and a minimum of 50,000 events were acquired for each sample. Detailed dot blot analysis was performed on both the stained tubes.
After excluding doublets, cell populations were analyzed on CD45-side scatter population. With the help of the CD33-SideScatter plot, granulocytes and monocytes were gated separately and were analyzed further. Granulocytes were analyzed in the CD24- FLAER (fluorescein-labelled proaerolysin) plot and monocytes were analyzed in the CD14-FLAER plot. The presence of a PNH clone was defined when more than one per cent population was seen in the double-negative quadrants for both granulocytes and monocytes.
Before starting the testing facility, peripheral blood samples from ten normal healthy controls were processed. In these controls, PNH clone (more than 1.0% in size) was not found. At least 50,000 events were acquired for these control cases. Hence, a 50 cell cluster was fixed as a minimum to identify the PNH clone which aided in labeling cases with >0.1 per cent and <1 per cent as small PNH clone. Based on this premise, we did observe a double-negative clone size around 0.02-0.04 per cent in monocytes or granulocytes of some of these ten controls. However, in none of the healthy controls, the clone size crossed 0.05 per cent.
Results & Discussion
Among the 61 cases of DVT recorded, the mean age was 45.21 yr (range: 21-87 yr). Of all the cases, 37 (61%) were male and 24 (39%) were female. All of the patients were from western Rajasthan.
The patients presented with thrombosis at different sites, as shown in Table I . The most common site was found to be the left lower limb, seen in 26 cases (43%), followed by the right lower limb seen in 10 cases (16%). The increased incidence of left lower limb DVT over right lower limb DVT in the present study is in concordance with the existing literature and is hypothesized to be due to compression of the left common iliac vein by the right common iliac artery, leading to stasis of blood10 , 11
Table I: Different sites of deep vein thrombosis among patients included in our study
Among the 61 cases of DVT, three cases (4.92%) revealed large clones (size of double-negative population >1%) on monocytes as well as granulocytes, hence fulfilling the criteria for diagnosing PNH, which is, the absence of at least two distinct GPI-anchored proteins, from two different cell lineages9 Table II and Fig. 1 ). One of these three cases also had pancytopenia, while the other two had isolated anaemia. Two of the three also had evidence of haemolysis. Since patient A3 showed pancytopenia, hence bone marrow examination was done wherein cellular bone marrow revealed all the haematopoietic components with marked erythroid prominence. Further, three patients (4.92%) showed small clones (size of double-negative population >0.1%, but <1%) on both granulocytes and monocytes (Table II and Fig. 2 ). Nine (15%) patients showed small clones only on granulocytes, and 11 (18%) patients showed small clones only on monocytes.
Table II: Details of cases in which large* (A1-A3) or small** (B1-B3) clone size was detected
Fig. 1: Scatter plot of patient A1: (A ) FLAER and CD24 on granulocytes, (B ) FLAER and CD 14 on monocytes, and (C ) RBC histogram for expression of CD59 on CD235 a positive RBC. FLAER, fluorescein-labelled proaerolysin; CD, cluster of differentiation; RBC, red blood cells
Fig. 2: Scatter plot of patient B1: FLAER and CD24 on granulocytes (A ), FLAER and CD 14 on monocytes (B ) and RBC histogram for expression of CD59 on CD235a positive RBC (C ). FLAER, fluorescein-labelled proaerolysin; CD, cluster of differentiation; RBC, red blood cells.
The three cases having large clones on granulocytes and monocytes were also tested for other thrombophilia markers that are protein C, protein S, activated protein C resistance, lupus anticoagulant, anticardiolipin antibody, anti-beta-2 glycoprotein antibody, fibrinogen and antithrombin. However, none of the above patients showed positivity for any of these markers.
Previous studies have associated the risk of thrombosis with PNH clone size <50 per cent6 12 thrombosis with small clone size <1 per cent. Moreover, thrombophilia is more commonly associated with the presence of haemolysis12 thrombosis . This could be reflective of the existence of small clones of PNH in the general population, the exact prevalence of which, however, is unknown5
Only a few studies worldwide have used FLAER-based markers to screen for PNH in patients presenting with thrombosis : A Canadian study conducted among patients with idiopathic venous thromboembolism showed a PNH clone size of >0.02 per cent in the neutrophil population in only one out of 388 patients5 thrombosis of the splanchnic vein showed small PNH clones (<0.2%) in only two out of 202 patients13 thrombosis as having large PNH clones14 thrombosis 15 thrombosis showed PNH clone size between 3.02 and 4.62 per cent in four out of 112 patients16
The present study in western Rajasthan shows a higher proportion of patients with PNH compared to those reported in studies previously done elsewhere5 , 13 - 16 thrombosis , if other indications such as coexistent cytopenias, or intravascular haemolysis are noted, or if the patients are young. This may provide an otherwise missed opportunity to diagnose this disorder. Patients with DVT are given anticoagulants17 18 18 19 thrombosis in PNH, and to determine its epidemiological aspects in the western Rajasthan region.
Acknowledgment : The authors acknowledge Dr Gopal Krishana Bohra, Associate Professor, department of Medicine, All India Institute of Medical Sciences, Jodhpur, who helped in enrolling patients, and Ms Jagriti Jha, MBBS student, AIIMS Jodhpur, who helped in sample collection.
Financial support & sponsorship : The first author (SP) received ICMR- Short Term Studentship (2019-05492) for conduction of this study.
Conflicts of Interest : None.
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