factor x , also known as stuart - prower factor , is a vitamin k - dependent serine protease produced in the liver . activated factor x cleaves prothrombin to thrombin , which facilitates the conversion of fibrinogen to fibrin which ultimately leads to the formation of a fibrin clot 1 . acquired factor x deficiency is commonly caused by the use of coumarin derivatives or due to liver disease isolated factor x deficiency is a rare disorder and was first described in the early 1950s 2,3 . only a few families have been described , probably due to factor x s essential role in the hemostasis 4,5 . symptoms vary from mild hemorrhage , such as bruising and mucocutaneous bleeding , to more severe bleeding , for example , gastrointestinal bleeding and muscle bleeding . bleeding severity depends on the plasma concentration of factor x. values between 1 and 10% are associated with minor bleeding or more serious bleeding provoked by surgical procedures or trauma , while spontaneous and severe bleeding is described with values below 1% 1,6 . acquired isolated factor x deficiency is mostly associated with amyloid light - chain ( al)-amyloidosis 713 . in amyloidosis , factor x binds permanently to amyloid fibrils within the vasculature , liver and spleen and is thereby scavenged from blood circulation 14 . there are also a few reports of factor x deficiency concomitant with respiratory disease , antibiotic treatment , leprosy , burns or malignancy such as acute myeloid leukemia 1528 . in some cases ( 1994 ) , proposed that in the context of post - infectious disease , similar epitopes may be found on viral antigens leading to generation of an anti - factor x inhibitor 24 . in several other cases the etiology of the acquired factor x deficiency remains unclear 17,19,21,23,27,28 in addition to earlier reports , we describe an 81-year - old patient without a bleeding history who presented with moderate to serious spontaneous bleeding symptoms due to clearance of factor x by a noninhibitory antibody . plasma for further testing was stored at 80c . the prothrombin time was measured using innovin reagent ( siemens healthcare , erlangen germany ) and the aptt using actin fsl reagent ( siemens healthcare ) . mixing studies were done by mixing equal volumes of patients plasma and normal plasma pools ( control n , siemens healthcare and in - house prepared pool ) . the aptt was measured immediately and after 2  h of incubation at 37c to detect a time dependent inhibitor . heparin was neutralized in 1  ml of plasma by addition of heparinase i using dade hepzyme ( siemens healthcare ) according to manufacturer s instructions . all factor assays were performed using one stage assay and factor x antigen level was determined using enzyme linked immunoassay ( elisa ) . for lupus anticoagulant testing a diluted russell s viper venom time ( la screen / mixing reagent , gradipore hawthorne ny ) and a lupus sensitive aptt ( biomerieux , marcy letoile france ) were used . the bethesda inhibitor assay for detecting inhibitory antibodies directed toward factor in - house prepared pooled plasma was used as normal plasma source ( a pool of more than 32 adult donors ) . radioimmunoassay ( ria ) for detecting factor x antibody was performed according to wolbink et  al . mg sepharose - immobilized protein a beads ( ge healthcare , little chalfont , buckinghamshire , uk ) were incubated overnight with 50  l prediluted in phosphate - buffered saline ( pbs)/0.3% bovine serum albumin ( bsa ; emd millipore , billerica , ma ) plasma of the patient , polyclonal rabbit antihuman factor x antibody or plasma from a healthy control in a total volume of 800  l pbs - at ( pbs containing 0.3% bsa , 0.2% tween-20 ( merck , darmstadt , germany ) and 0.01  mol / l ethylenediamine tetraacetic acid ( edta ) , thereby coupling all antibodies to the sepharose beads . the beads were subsequently washed two times with pbs - t ( pbs containing 0.005% tween-20 ) and four times with tris - buffered saline ( tbs)-ct ( 10  mmol / l tris , 5  mmol / l cacl2 , 140  mmol / l nacl , ph7,4 with 0.05% tween-20 ) . beads were resuspended in 0.5  ml tbs - c - at ( tbs - c containing nan3 , bsa and 0.2% tween-20 ) . thereafter beads were resuspended in 0.5  ml tbs - c - at and 2  ng of biotin labeled factor x ( provided kindly by m. boon - spijker , dept . plasma protein , sanquin research , amsterdam ) was added to the samples followed by overnight incubation and rotation using a rotor . thereafter the beads were washed five times with tbs - ct and incubated overnight with 50  l radioactive i labeled streptavidin dialyzed in tbs - ca and diluted in 500  l tbs - c - at . unbound i was removed by washing five times with tbs - ct and samples were counted using a wallac 1260 multigamma ii counter . the bethesda inhibitor assay for detecting inhibitory antibodies directed toward factor x was done according to the classical bethesda assay described for factor viii 29 . in - house prepared pooled plasma was used as normal plasma source ( a pool of more than 32 adult donors ) . factor radioimmunoassay ( ria ) for detecting factor x antibody was performed according to wolbink et  al . mg sepharose - immobilized protein a beads ( ge healthcare , little chalfont , buckinghamshire , uk ) were incubated overnight with 50  l prediluted in phosphate - buffered saline ( pbs)/0.3% bovine serum albumin ( bsa ; emd millipore , billerica , ma ) plasma of the patient , polyclonal rabbit antihuman factor x antibody or plasma from a healthy control in a total volume of 800  l pbs - at ( pbs containing 0.3% bsa , 0.2% tween-20 ( merck , darmstadt , germany ) and 0.01  mol / l ethylenediamine tetraacetic acid ( edta ) , thereby coupling all antibodies to the sepharose beads . the beads were subsequently washed two times with pbs - t ( pbs containing 0.005% tween-20 ) and four times with tris - buffered saline ( tbs)-ct ( 10  mmol / l tris , 5  mmol / l cacl2 , 140  mmol / l nacl , ph7,4 with 0.05% tween-20 ) . beads were resuspended in 0.5  ml tbs - c - at ( tbs - c containing nan3 , bsa and 0.2% tween-20 ) . thereafter beads were resuspended in 0.5  ml tbs - c - at and 2  ng of biotin labeled factor x ( provided kindly by m. boon - spijker , dept . plasma protein , sanquin research , amsterdam ) was added to the samples followed by overnight incubation and rotation using a rotor . thereafter the beads were washed five times with tbs - ct and incubated overnight with 50  l radioactive i labeled streptavidin dialyzed in tbs - ca and diluted in 500  l tbs - c - at . unbound i was removed by washing five times with tbs - ct and samples were counted using a wallac 1260 multigamma ii counter . an 81-year - old previously healthy caucasian man presented on the emergency ward with a spontaneous hematoma of the skin and soft tissue in his left abdominal flank and around his left shoulder . two weeks prior to admission , he had received a red blood cell concentrate transfusion after an episode of acute anemia caused by bleeding due to a minor trauma . complaints consisting of tiredness and slight weight loss in the past month were reported upon admission . he did not use any anti - platelet drugs or anticoagulants , which was confirmed by toxicology screening . at presentation , on the left side of his abdomen there was a painful hematoma of approximately 12    8 inches . a painless swelling of about 8    8 inches was present on his left shoulder region . computed tomography ( ct ) of thorax and abdomen showed intramuscular hematoma of the latissimus dorsi on the left side and enlarged inguinal , intra - abdominal and intrathoracal lymph nodes . initial laboratory evaluation revealed anemia with a hemoglobin level of 7.1  g / dl ( 12.916.9  g / dl ) , with normal red blood cell indices . however , lactate dehydrogenase ( ld ) was slightly elevated : 325  u / l ( < 248 further analysis showed neither serum m - protein by electrophoresis nor elevation of serum - free light chains . both , prothrombin time ( pt ) and activated partial thromboplastin time ( aptt ) were prolonged 23.3  sec ( pt normal range 912  sec ) and 51  sec ( aptt normal range 2434  sec ) , respectively . pre - analytical contamination of the sample with heparin was ruled out using hepzyme treatment . the in  vitro addition of normal plasma to the patients plasma resulted in complete correction of pt and aptt times , immediately after mixing as well as after 2  h of incubation at 37c . this result is suggestive of a factor deficiency rather than an antibody neutralizing procoagulant function . in the acute clinical situation the patient initially received three red blood cell concentrates resulting in an increase of his hemoglobin level to 9.3  g / dl . treatment with vitamin k , prednisone , fresh frozen plasma ( ffp ) and prothrombin complex concentrate ( pcc ) was started , which resulted in a temporary and partial response on the clotting times ( fig.1a ) . more detailed analysis of blood coagulation factor levels showed slightly reduced activity of factor ii ( 54% ) , vii ( 52% ) , ix ( 56% ) and xii ( 64% ) and extremely low activity of factor x ( 34% ) ( table1 ) . the factor x activity was measured both via the intrinsic and extrinsic pathway . additionally , a markedly reduced factor x concentration was found with the antigen assay correlating with the functional deficiency . blood coagulation parameters at initial laboratory screening and before treatment pt , prothrombin time ; aptt , activated partial thromboplastin time . ( a ) effect on activated partial thromboplastin time ( aptt , dashed lines ) and prothrombin time ( pt , solid lines ) in response to fresh frozen plasma ( ffp ) , vitamin k and prothrombin complex concentrate ( pcc ) in first 10  days of hospitalization of acute setting . each green triangle represents one unit of ffp , 5  mg of vitamin k , 80  mg prednisone or 500  u pcc . the factor x deficiency in our patient was considered to be acquired , given the absence of a bleeding history . in an attempt to clarify the cause of the acquired factor x deficiency , the patient underwent a biopsy of an enlarged inguinal lymph node . furthermore , neither bone marrow biopsy examination nor the aspirate from the abdominal fat showed signs of amyloidosis . presence of a factor x antibody was suspected based on the lack of response to ffp and pcc . we were not able to detect an inhibitor in the mixing studies or using a bethesda assay against functional antibodies inhibiting factor x activity . despite the fact that there was no inhibitory antibody detectable , the probability of the presence of a noninhibitory antibody was considered and consequently the patient was first treated with prednisone as first - line treatment for an auto - immune antibody - mediated disease . because of a new abdominal soft tissue bleeding , more rigorous treatment was initiated in an attempt to firmly tackle the marginal - zone lymphoma , considering a relation between the factor x deficiency and the lymphoma . treatment consisted of chemo- and immunotherapy , chlorambucil and rituximab , resulting in normalization of pt , aptt levels ( 10.7 and 28  sec , respectively , fig.1b ) , ldh level of 210 the normalization of the factor x activity after treatment of the patient strongly suggested that a noninhibitory antibody against factor x was present . such an antibody would bind factor x without inhibiting the function but facilitating fast clearance from the circulation . laboratory evaluation of vitamin k dependent procoagulant factor activities just before and after rituximab and chlorambucil treatment are shown in table2 . a ria assay detecting factor x antibody binding capacity was used to demonstrate the presence of a possible factor x antibody in the patient plasma before treatment and after normalization . indeed , the presence of a noninhibitory antibody was ultimately demonstrated in the before treatment sample of our patient and not in the sample after treatment ( table3 ) . as a positive control rabbit antihuman factor x the patient samples were measured in a 1:50 and 1:250 dilution , both in duplicate . response curve of our positive control , showing a low but detectable amount of factor x binding . plasma from a healthy person and patient plasma after treatment were used as negative controls , in this experiment . in both samples no signal above the blank was observed . no binding of the antibody to biotinylated factor x in the absence of calcium was observed ( data not shown ) . vitamin k dependent procoagulant clotting factor activities before and after rituximab and chlorambucil treatment bt , before treatment ; at , after treatment . results ria assay before and after rituximab and chlorambucil treatment bt , before treatment ; at , after treatment . serial dilution of positive control rabbit anti - human factor x antibody was made and incubated overnight with sepharose a beads to capture them . after washing , biotin labeled factor x was added and incubated with the beads overnight . the amount of i - streptavidin bound was measured after washing and calculated back to the percentage of biotin binding to the antibody . factor ii , vii and ix were also slightly reduced in our patient , although to a much lesser extent than factor x. no binding antibody could be detected for factor ii and ix using the same ria experiment using biotinylated factor ii and ix as antigen , suggesting a specific antibody against factor x only . one year after the diagnosis , the patient remains asymptomatic with normal factor x levels . a ct - scan to evaluate effect of chlorambucil and we described a previously healthy 81-year - old man with an extensive and spontaneous cutaneous- and soft tissue hematoma as well as muscle bleeding associated with a nodal marginal - zone lymphoma . since our patient did not have a bleeding tendency in the past , we concluded that this patient suffered from an acquired factor x deficiency . acquired factor x deficiencies are most frequently described in relation to amyloidosis , factor x inhibitors or vitamin k deficiency 7,16 . at the first instance , because all measured vitamin k - dependent clotting factors were reduced , it was thought that the patient had a mild vitamin k deficiency . however , it is rather unlikely that vitamin k deficiency was the main cause of the grossly reduced factor x activity and therefore the cause of the bleeding complication . moreover , this assumption is in agreement with the nonresponsiveness observed after vitamin k supplementation . this is an indolent disease and treatment is initiated only in the presence of symptoms . considering the physiology of the marginal zone in lymph nodes , where mature b cells can rapidly proliferate and differentiate into antibody secreting plasma cells , it was hypothesized that an antibody related factor x deficiency was present . initial treatment with ffp or pcc demonstrated a temporary response in the factor x levels . the period of response did not correlate with the half - life of factor x , which is around 4872  h 31 . this result suggested the presence of an antibody causing enhanced clearance of factor x from the blood circulation . the patient had combined deficiency of factor ii and x. a cross - reactive antibody was found to bind to factor ii , ix and x via the common metal - ion - dependent conformational epitope found on the vitamin k - dependent -carboxyglutamic acid ( gla ) domain 27 . also , in a patient with a low grade follicular center cell non - hodgkin lymphoma , the presence of a noninhibitory autoantibody binding via a calcium - dependent epitope of factor ii was found , which accelerated its clearance 32 . in an another patient also a calcium - dependent noninhibitory antibody to factor ii was found , which had low affinity for factor ii 33 . the presence of a noninhibitory antibody against factor x was found in our patient , which was calcium dependent . this suggests that our antibody is directed against a factor x epitope that is metal dependent , maybe also the common metal - ion - dependent conformational epitope found on the gla domain . in the ria assay using biotinylated factor ii and ix , we could not detect binding to these clotting factors . due to the severity and recurrence of bleeding we had promptly started treatment with rituximab and chlorambucil according to the guidelines for treatment of nodular marginal - zone lymphoma 34 . treatment resulted in complete remission of the lymphadenopathy and normalization of factor x levels , proving that factor x deficiency was related with the non - hodgkin lymphoma . , reported a patient with an acquired von willebrand disease related to a marginal - zone lymphoma 35 . the authors suggest that the von willebrand factor ( vwf ) protein was absorbed by tumor cells , showing an aberrant tumor - cell expression of the platelet vwf receptor ( gbib ) . another case report in which a patient was diagnosed with splenic marginal - zone lymphoma without bleeding problems , showed reduced factor activities of factor ii , v , vii , viii , ix , x , xi and xii . the authors found a specific inhibitor against factor ix and assumed presence of a non - factor specific inhibitory antibody 36 . our case is another example of an acquired factor x deficiency associated with the presence of a lymphoproliferative disease . this is the first patient to our knowledge diagnosed with an acquired factor x deficiency caused by a noninhibitory antibody related to nodal marginal - zone lymphoma . we advise to start treatment of the underlying disease as soon as possible when there are symptoms of hemorrhage or if there is a high risk of hemorrhage due to low - factor x levels .
key clinical messageprolonged clotting times were observed in a patient with spontaneous hemorrhage . 
 analysis showed severe factor x deficiency due to clearance by a noninhibitory antibody . 
 lymphadenopathy identified on imaging led to diagnosis of marginal b - cell lymphoma . 
 treatment of lymphoma with rituximab and chlorambucil resulted in complete disappearance of the bleeding disorder .