Haematological parameters and pathological clotting in deep vein thrombosis and patients with HIV

Background. Patients infected with the human immunodeficiency virus (HIV) are more prone to systemic inflammation and pathological clotting, and many may develop deep vein thrombosis (DVT) as a result of this dysregulated inflammatory profile. Coagulation tests are not routinely performed unless there is a specific reason. Methods. We recruited ten healthy control subjects, 35 HIV negative patients with deep vein thrombosis (HIV negative-DVT), and 13 HIV patients with DVT (HIV positive-DVT) on the primary antiretroviral therapy (ARV) regimen- Emtricitabine, Tenofovir and Efavirenz. Serum inflammatory markers, haematological results, viscoelastic properties (using thromboelastography-TEG) and scanning electron microscopy (SEM) of whole blood (WB) were used to compare the groups. Results. DVT patients (HIV positive and HIV negative) have anaemia with raised inflammatory markers which are more pronounced in HIV positive patients. HIV positive-DVT patients also have a microcytic hypochromic anaemia. DVT patients have a hypercoagulable profile on the TEG but no significant difference between HIV negative-DVT and HIV positive-DVT groups. The TEG analysis compared well and supported our ultrastructural results. Scanning electron microscopy of HIV positive-DVT patient’s red blood cells (RBCs) and platelets demonstrates inflammatory changes including abnormal cell shapes, irregular membranes and microparticle formation. All the ultrastructural changes were more prominent in the HIV positive-DVT patients. Conclusions. HIV positive patients have an increased hypercoagulability and DVT prevalence. Our results point to the importance of looking at the coagulation system Hct= haematocrit, MCV= mean corpuscular volume, MCH=mean corpuscular haemoglobin, MCHC=mean corpuscular haemoglobin concentration, RCDW=red cell distribution width, Plt=platelet count, MPV=mean platelet volume.

(http://www.unaids.org/en/resources/fact-sheet). Although treatment of the infection with antiretroviral regimes (ARVs) is essential to addressing the pandemic, the condition is characterized by a large plethora of additional conditions associated, and also co-existing with the HIV infection, including the presence of systemic inflammation. Systemic inflammation is associated with an increase in circulating pro-inflammatory biomarkers, and is closely associated with an amplified propensity to form pathological blood clots (which is associated with hypercoagulability or an over-activated coagulation system) 2-8 .
During HIV infection, various circulating inflammatory biomarkers, including cytokines interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF)-α and also other pro-inflammatory biomarkers are present 9 . An increase in these biomarkers are also present in cardiovascular disease 10,11 ; and it is therefore not surprizing that HIV positive individuals are known to have an increased presence of cardiovascular complications 12,13 , including an increased risk to develop atherosclerosis and venous thromboembolic disease 14 ; and also DVT [15][16][17] . The presence of DVT is also classified as a systemic inflammatory process 18 , and associated with pathological clotting and upregulated circulating inflammatory biomarkers 19 .
The prevalence of developing a DVT in HIV positive individuals is increased two to ten times compared to the general population 20 . HIV positive individuals also have a 43% increase in age-adjusted odds ratio for pulmonary embolism, a common complication of DVT, compared to HIV negative individuals 21 . Multiple coagulation abnormalities have been reported in HIV positive patients such as decreased levels of protein C and S; and increased levels of von Willebrand factor 22-24 . However, the association of these abnormalities with DVT is not always consistent 22,25 .
Coagulation investigations are therefore not performed routinely in patients with HIV infection. Standard coagulation investigations are also not performed routinely as part of the management in patients with DVT, with the exception of a D-dimer which is used to assist with the diagnosis 26-28 .
In the current paper, we therefore study the haematological profiles, including clotting and various inflammatory markers, in the presence of DVT in HIV positive and HIV negative individuals and compare the results to that of healthy individuals.
We compare inflammatory markers for iron (iron saturation, transferrin and serum ferritin), fibrinogen, high-sensitive C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and haematology analyser results, together with viscoelastic properties of whole blood (WB) and platelet poor plasma (PPP). We also looked at ultrastructure of platelets and erythrocytes/red blood cells (RBCs) (using whole blood smears) with the SEM, as well as after thrombin was added to whole blood, to study clot structure.

materials and methods
The aim was to compare the inflammatory and haematological profile of HIV patients with DVT to HIV negative patients with DVT. An analytical and descriptive prospective case control study was used from 2 Pretoria academic hospitals,

Inflammatory marker analysis
Serum iron (total iron in blood) levels were measured together with iron saturation, transferrin (iron binding protein) and serum ferritin (iron storage form). Serum iron levels were measured by a modification of the automated AAII-25 colorimetric method. Fibrinogen (quantitative measurement of functional fibrinogen by automated coagulation analysers), CRP (measured by latex-enhanced nephelometry) and ESR (measured by an automated ESR analyser) levels were also assessed.

Haematology analysis
A haematology analyser (Advia 2120i, Siemens Healthcare) was used to do full blood counts, and the analysis included white cell count (and its differential count), RBC count, haemoglobin level, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC), as well as platelet count and mean platelet volume (MPV).
Viscoelastic properties of whole blood and platelet poor plasma using thromboelastography Citrated WB, as well as PPP were used. Whole blood, collected in a citrate tube, was centrifuged to obtain PPP (15 minutes at 3000g). Whole blood was used to assess the full coagulation process, while PPP was used to assess coagulation without the influence of platelets and RBCs on the viscoelastic properties of the clot. Calcium chloride was added to either WB or PPP and 7 different parameters measured, which included: reaction time (R-time), kinetic time (K-time), alpha angle, maximum amplitude (MA), maximum rate of thrombus generation (MRTG), time to maximum rate of thrombus generation (TMRTG) and total thrombus generation (TTG).

Ultrastructure of platelets and red blood cells (RBCs)
The ultrastructure of platelets and RBCs were studied after preparing whole blood smears for scanning electron microscopy (SEM). 10 µl of WB was placed directly on a glass microscope slide, followed by fixing in 2.5% glutaraldehyde, dehydration (as per usual SEM preparation) 29 and mounting. Micrographs were taken with Zeiss Crossbeam 540 Field Emission Gun Scanning Electron Microscopy.

Statistical analysis
Graphpad 5 was used to do one-way ANOVA analysis. A post-test to compare groups was performed using Tukey's multiple comparison test.

Results
Inflammatory marker and haematological parameter analysis Inflammatory marker analyses are shown in Table 1 and haematology analysis are shown in Table 2. Both HIV negative-DVT and HIV positive-DVT groups have anaemia. The HIV negative-DVT group appears to have anaemia due to inflammation (this type of anaemia is typically found in chronic disorders), whereas the anaemia in the HIV positive-DVT group is rather due to iron deficiency. The changes in serum iron, transferrin and ferritin in the HIV positive-DVT group reflects low systemic iron status, but the raised serum ferritin (although not statistically significant) may be due to the inflammatory status of the individuals.
Inflammation is reflected, whether from the DVT or the HIV infection, by the raised white cell count, fibrinogen concentration, CRP and ESR. Surprisingly, the platelet count was not decreased in the HIV positive-DVT group. We expected this parameter, as well as the MPV to be markedly decreased, due to e.g. HIV thrombocytopaenia, which is usually common amongst HIV patients, but in our sample this was not the case.  Interestingly, there were no significant differences in hypercoagulability between the HIV positive-DVT and the HIV negative-DVT groups.     The RCDW is the coefficient of variation of RBC volume. The higher the value, the more anisocytosis (unequal RBC sizes) present. The RCDW of the HIV positive-DVT group was greater than the control and HIV negative-DVT groups ( Table 2). A raised RCDW is commonly associated with a decrease in haemoglobin and MCV concentration; but with a raised CRP, fibrinogen and white cell count 43 . This correlates with the haematological and inflammatory markers found in the HIV positive-DVT group (Table 1 and 3). RCDW is strongly associated with mortality.
Patel and colleagues reported the all-cause mortality risk increases by 22% for every 1% increase in RCDW. Furthermore, the physiological association between RCDW and mortality has been hypothesised to be related to the systemic factors involved in inflammatory conditions and oxidative stress which affects erythrocyte maturation and degradation 43-45 .
The ESR is the extent in which erythrocytes sediment in one hour 46 . The ESR in both the HIV negative-DVT and HIV positive-DVT groups were raised compared to the control group (Table 1). In inflammatory conditions the ESR rises as the erythrocytes become sticky and adhere to each other which can be seen as rouleaux formation [47][48][49] .
Fibrinogen, a high molecular weight plasma protein, is a crucial factor in the coagulation pathway (factor I). Increased fibrinogen levels are associated with thrombo-embolic events. Fibrinogen also has a role in inflammation as it tends to adhere to the membrane receptors of cells involved with inflammation. Fibrinogen can adhere to the RBCs, which becomes "heavier" resulting in an increased ESR and blood viscosity 50-57 . The fibrinogen levels were greater (but not statistically significant) in the HIV negative-DVT and HIV positive-DVT groups compared to the control group (Table 1)  Iron deficiency appears to be the main contributor to anaemia in the HIV positive-DVT group in keeping with a microcytic (low MCV), hypochromic (low MCH) anaemia (Table 2) 76,77 . A low serum iron level, low transferrin level and a low iron saturation percentage, but with a raised ferritin level seen in the HIV positive-DVT group, can be explained by an immunologically altered iron metabolism where the body has adequate or increased iron stores but is unable to utilize those stores 34,39,62,63,73,[78][79][80] . This functional iron deficiency can be considered a host defence mechanism by withholding iron from possible pathogens 81,82 . However, as iron is required for normal immune function, iron deficiency can also increase the risk of infection 82 .
Although the inflammatory RBC changes have been documented in noncommunicable diseases, there are only a few reports of communicable diseases, specifically HIV, and the effect on RBCs and the coagulation system 83-88 . Multiple abnormal RBC shape changes and membrane abnormalities were noted in the patients with DVT (HIV negative and HIV positive groups) (Figure 1 to 3). During inflammatory diseases, RBCs exposed to oxidative stress and inflammatory molecules undergoes biochemical membrane changes which can result in biophysical shape changes and eryptotic cells [89][90][91][92][93][94][95][96][97] . Eryptosis is a co-ordinated suicidal death of the red blood cells, similar to apoptosis, that allows for the removal of defective, infected or potentially harmful cells before they undergo haemolysis [98][99][100][101][102] . The abnormal RBCs present with an abnormal expression of phosphatidylserine, a cell membrane lipid, on the external membrane layer. RBCs that display phosphatidylserine also contribute to the hypercoagulation state and they provide a prothrombotic surface for the formation of thrombin 42,100,103-114 .
Membrane vesicle formation and microparticle shedding (microscopic extracellular membranous structures) were also seen in both DVT groups. RBC-derived microvesicles or microparticles, is known to be associated with the expression of phosphatidylserine 115 . RBC-derived microparticles appear to enhance thrombin generation resulting in a hypercoagulable state, such as in post transfusion DVT, sickle cell disease and haemolytic anaemia 116,117 . As the microparticle presence might also be associated with increased thrombin presence, the complement system can therefore also be activated and thereby enhance the systemic inflammatory response which is also a hypercoagulable state 118 . Microparticles are also thought to originate from CD4 lymphocytes 119  This can happen as part of the HIV and DVT pathology. The (hyper) activation of platelets, together with an abnormal matted fibrin network, contracts the clot containing the trapped pathological RBCs into a tight package (Figure 3B and 3C).
The result is the formation of polyhedrocytes, which is commonly found in DVT 123 .
Platelet functioning depends on the quality and the quantity of the platelets 124 .
Platelet count is a measure of the number of platelets in a volume of blood.
Thrombocytopenia (low platelet count) is a common finding in HIV positive patients, whether it be due to increased destruction or decrease production of platelet cells 58 . However, in this study both the HIV negative-DVT and HIV positive-DVT groups had a non-statistically significant increase in the platelet count ( Table 2) Our TEG analysis compared well and supported our ultrastructural results ( Table 3).
The HIV negative-DVT group compared to the control group showed significant differences with regards to TMRTG, while the HIV positive-DVT group compared to the control group, showed significant differences with regards to R-time, and the TMRTG. According to Pretorius and colleagues not all the parameters need be abnormal to indicate pathological coagulability and the degree of coagulability can be related to the number of parameters that are abnormal 5 .
Both DVT groups (HIV negative and HIV positive), using whole blood, indicate a hypercoagulable profile that has a rapid initiation and amplification, resulting in the rapid formation of thrombin. The TEG parameters in the HIV positive-DVT group compared to the HIV negative-DVT group indicate a hypercoagulable profile but there were no statistical significance in any of the parameters. In the HIV negative-DVT and HIV positive-DVT groups, both having rapid R-times and K-times (Table 3), trapped RBCs between a matted (hypercoagulable) fibrin network were noted.
Considering that pulmonary embolism can result in up to 10% mortality 139 Figure 1 Scanning electron microscopy micrographs of (A) representative healthy RBCs, (B) a represe Representative scanning electron microscopy micrographs of RBCs and platelets from HIV po