Lymphocytosis in Adults

Normal Peripheral Blood Lymphocytes

Lymphocytes are small leucocytes with a round nucleus and a thin rim of cytoplasm. About 10% of the peripheral lymphocytes are large, have a more abundant cytoplasm that has granules. These are called large granular lymphocytes (LGL). About 85% of the peripheral blood lymphocytes are T lymphocytes. The remaining are B lymphocytes and NK cells with B lymphocytes dominating.

Lymphocytosis is increase in the lymphocyte counts to what is normal for age. Lymphocyte count is highest at birth and falls with age. Normal lymphocyte counts in adults is 1000 to 4800 cells/mm3.

Causes of Lymphocytosis

Lymphocytosis may be reactive (see table below) or due to a malignancy. Reactive lymphocytosis is polyclonal. Lymphocytosis due to malignancy is monoclonal. The most common cause of reactive lymphocytosis is infectious mononucleosis due to Epstein-Barr virus infection. Reactive lymphocytosis may be seen in other viral infections, drug hypersensitivity, thymoma and after splenectomy. Persistent polyclonal B cell lymphocytosis is a condition found in middle aged women who smoke.

Monoclonal B cell lymphocytosis and chronic lymphocytic leukaemia are the commonest causes of primary lymphocytosis.

Causes of Lymphocytosis
Reactive lymphocytosis (reactive lymphocytes unless mentioned otherwise)

  1. Infections
    1. Viral: Infectious mononucleosis (EBV), Adenovirus, CMV, Coxsackie virus, Hepatitis, Acute HIV infection, Human T-lymphotropic virus type I, Influenza, Measles, Mumps, Poliovirus, Rubella
    2. Bacterial: Pertussis, Cat scratch disease and other chronic bacterial infections
    3. Parasitic: Toxoplasmosis
  2. Drug hypersensitivity reactions
  3. Stress (normal looking lymphocytes)
  4. Persistent polyclonal B cell lymphocytosis (lymphocytes have a distinct nuclear cleft)
  5. Post-splenectomy  (normal looking lymphocytes)
  6. Thymoma  (normal looking lymphocytes)
  7. Hyperreactive malarial splenomegaly  (normal looking lymphocytes)

Primary Lymphocytosis

  1. B Lymphocytic
    1. Monoclonal B cell lymphocytosis
    2. Chronic lymphocytic leukemia
    3. B-Prolympocytic leukaemia
  2. Leukaemia Phase of non-Hodgkin lymphoma (common
    1. B cell Follicular lymphoma
    2. Mantle cell lymphoma
    3. Splenic marginal zone lymphoma
    4. Lymphoplasmacytic lymphoma
  3. T Cell Lymphoma
    1. Prolymphocytic leukaemia
    2. Sezary cell leukaemia
    3. Adult T-cell leukaemia/lymphoma
    4. Large Granular cell leukaemia
  4. Acute lymphoblastic leukemia

Clinical Profile of Patients with Lymphocytosis

Infectious Mononucleosis: Infectious mononucleosis (IM) is a self limiting disease of adolescents that presents with prodrome of fatigue, myalgia lasting 1-2 followed by fever pharyngitis and lymphadenopathy, splenomegaly, hepatomegaly and rash. The peripheral smear shows lymphocytosis with atypical lymphocytes. These are irregular, have slightly larger nuclei, a more open chromatin and abundant cytoplasm. Some cells may appear blastoid. Atypical lymphocytes are predominantly CD8+. Differentiation from lymphocytosis of malignancy is made by

  1. Clinical Picture: Patients with IM have fever, malaise, pharyngitis and other features of IM.
  2. Morphology of Lymphocytes: Reactive lymphocytes of IM show variations in size and morphology. Cells from patients with malignant lymphocytoisis are more uniform.

Other infection: Atypical lymphocytosis is a feature of other infections listed in the table above. Typical features of the causative infection may or may not be present. CMV associated mononucleosis syndrome is indistinguishable from IM. Lobulated lymphocytes are a feature of human T-lymphotropic virus type I (HTLV-I ) infection.

Lympocytosis with B pertussis: Pertusis caused by Bordetella pertussis with upper respiratory symptoms that evolves into a paroxysmal cough in about 1-2 weeks. Pertusis is an exception amongst acute respiratory infections in that it causes lymphocytosis rather than neutrophilic leucocytosis. Unlike other viral infections lymphocytes of pertussis are small and have cleaved nucleus.

Stress Lymphocytosis: Stress has been associated with an increased lymphocyte count. Counts may range 4,000 to 10,400/mm3. All subsets of lymphocytes increase. The counts normalises after the stressful event passes (Am J Clin Pathol. 2002 May;117(5):819-25).

Persistant Polyclonal B cell lymphocytosis: Persistent polyclonal B cell lymphocytosis is seen in young to middle-aged women who are smokers. The patients show the presence of large binucleate lymphocytes. There is polyclonal increase in IgM in the serum. The patients do not have lymphadenopathy and splenomegaly.

Splenectomy: Lymphocytoisis that persists has been reported in patients who have undergone splenectomy (Clin Lab Haematol. 1995 Dec;17(4):335-7).

Thymoma: Paraneoplastic T cell lymphocytoss may rarely be seen in patients with thymoma. The patients present with a mediastinal mass and lymphjocytiosis. They need to be differentiated from T Lymphoblastic leukaemia who can present with mediastinal mass with lymphoblasts (Ann Oncol 2007; 18:603-604).

Hyperreactive malarial splenomegaly: Hyperreactive malarial splenomegaly is seen in residents of malarious areas. It presents with left upper quadrant pain, fatigue and dyspnoea. The patients have a massive spleen. The haemogram shows anaemia, leucopenia and thrombocytopenia. Some patients may have lymphocytosis.

Monoclonal B cell lymphnocytosis: Monoclonal B cell lymphocytosis is a premalignant condition that has a risk of progressing to chronic lymphocytic leukaemia. It it is diagnosed in a patients with lymphocyte count <5000/mm3 without any other evidence of lymphproliferative disease. The morphology and phenotype of the cells is identical to that of chronic lymphocytic leukaemia. Patients are asymptomatic and the disorder is detected incidentally on a haemogram performed for another reason.

Chronic lymphocytic Leukarmia (CLL)/small lymphatic leukaemia: Chronic lymphocytic leukaemia is the leukaemia phase of small lymphocytic lymphoma. Patients with CLL have lymphocytosis with normal looking lymphocytes. A few prolymphocytes may bee seen. If the percentage of prolympocytes is greater then 55% a diagnosis of prolymphocytic leukaemia should be made. The cells express CD19, CD20(usually weak), CD23 and the T cell marker CD5. Patients may have lymphadenopathy, splenomegaly and hepatomegaly. Anaemia and/or thrombocytopenia may co-exist, some of which may be due to autoimmunity.

Prolymphocytic Leukaemia: Prolymphocytic leukaemia is a misnomer. The malignant cell is actually a activated mature lymphocytes. Prolympnocytic leukaemia may be of B cell or T cell type. The cells are twice the size of a normal lymphocyte. The nucleus is round with a moderately condensed cytoplasm. A prominent central nucleolus is present. The cytoplasm is faintly basophilic. Morphiology of T Prolymphocytic leukaemia is similar. Patients present with a high count (usually > 100X109/L), massive splenomegaly in the absence of lymphadenopathy. T PLL may show skin infiltration in 20% of the cases. They may also show serous effusions.

Peripheral blood involvement with non-Hodgkin Lymphoma: Peripheral blood involvement with non-Hodgkin Lymphoma presents with mononucleated cells. Morphological features may  suggest the type of lymphoma. These include villous lymphocytes of splenic marginal zone lymphoma and atypical hairy cell leukaemia, cells with cribriform nuclei in Sézary syndrome plasmacytoiod lymphocytes of lymphplasmacytic lymphoma and lobulated lymphocytes in adult T-cell leukaemia/lymphoma. 

Large Granular Cell Leukaemia: LGL leukaemia is characterised by a count of 2-10X109/L. The cell are large granular lymphocytes with abundant cytoplasm and fine and coarse azurophilic granules. 



Anaemia with Hyperbilirubinaemia

A 49-year-old female presented with dyspnoea on exertion of 1 month duration. Examination reviled pallor and icterus. There was no lymphadenopathy, clubbing, koilonychia, platonychia, petechiae or purpura. There was no oedema of feet. The pulse was 90/min and the blood pressure 130/70 mm of Hg. Examination of the respiratory, cardiac and nervous systems did not show any abnormality. There was no organomegaly.

The haemoglobin was 4.9 g/dL with an erythrocyte count 1.37 x 1012/L, haematocrit of 16%, MCV of 116.78 fL, MCH of 35.77 pg and MCHC 30.63 of g/L.  The leucocytes count was 2800 with 35% neutrophils and 65% lymphocytes. The platelet count was 90 x 109/L. The peripheral smear showed macrocytosis and anisocytosis. Hypersegmented neutrophils were seen. The reticulocyte count was 3%.

The bilirubin was 2.1 mg/dL with a direct bilirubin of 1.8mg/dL and an indirect bilirubin of 0.3mg/dL. The Lactate dehydrogenase was 1417IU (normal 105 – 333 IU/L).

Anaemia and unconjugated hyperbilirubinaemia are characteristic of haemolysis. Does this patient have haemolytic anaemia?

Haemolysis shortens erythrocyte lifespan and results in increases haemoglobin breakdown. Haemoglobin is made of heme and globin. Heme consists of porphyrin ring at the centre of which is iron in the ferrous state. Iron released from catabolism of heme is reused. The porphyrin ring is catabolised to bilirubin. The bilirubin is transported to the liver for conjugation and excretion (see haemoglobin catabolism). Patients of haemolytic anaemia have unconjugated hyperbilirubinaemia because the increased bilirubin production overwhelms the hepatic bilirubin conjugation capacity.

One of the characteristics of megaloblastic anaemia is ineffective erythropoiesis. Ineffective erythropoiesis is defined as a sub-optimal (fewer) production of mature erythrocytes from a proliferating pool of immature erythroblasts. Each immature erythroblast produces less than the optimal number of erythrocytes because of premature death of erythroid precursors including haemoglobinized precursors. The haemoglobin released from haemoglobinized erythroid precursors is catabolised in the same manner as haemoglobin released from lysed erythrocytes (see haemoglobin catabolism). Megaloblastic anaemias are associated with unconjugated hyperbilirubinaemia because of death of haemoglobinized erythroid precursors.

The treatment of haemolytic anaemia and megaloblastic anaemia are different? How does one differentiate megaloblastic anaemia from that because of haemolytic anaemia? Does this patients have a haemolytic anaemia or megaloblastic anaemia?

Haemolytic anaemia is characterised by shortened erythrocyte survival. Erythrocytes survival is estimated by the use of radionucleotides something that is not possible at most centres. In clinical practice, a shortened erythrocyte survival is inferred from a high reticulocyte count. Reticulocytes are erythrocytes that have been produced in the preceding 24 hours. The erythrocytes survival is about 120 days and about 1% of erythrocytes are produced every day. Consistent with this the normal reticulocyte count is 0.5-1.5%.In patients of haemolytic anaemia, ddestruction of erythrocytes is matched by an increased production by the bone marrow. This manifests as reticulocytosis (see reticulocyte count). Megaloblastic anaemia occurs because of decreased production of erythrocytes and this manifests as reticulocytopenia. The difference between haemolytic anaemia and megaloblastic anaemia is the reticulocytosis in the former reticulocytopenia in the latter. This patient had a high reticulcoyte count but after correction both the reticulocyte production index [0.43] and corrected reticulocyte count [1.07%] were low excluding haemolysis. This patient was evaluated for megaloblastic anaemia.

The haemogram has clues to differentiate between haemolytic anaemia and megaloblastic anaemia. These include

  1. A very high MCV: The MCV is very high. Patients with haemolytic anaemia have a mild elevation in MCV. An MCV value >110fL is almost exclusively found in megaloblastic anaemias because of folate and/or B12 deficiency.
  2. Pancytopenia: B12 and folate deficiency impair DNA synthesis impairing erythrpoieis, myelopoiesis and megakaryopoiesis. Nutritional megaloblastic anaemias because of vitamin B12 and/or folate deficiency may show pancytopenia.
  3. Hypersegmented neutrophils (>5% neutrophils with >5lobes) is a feature of megaloblastic anaemia

Other features of megaloblastic anaemia include rise serum transferrin receptor, increased serum iron, serum ferritin and methemalbumin levels. Like haemolytic anaemia the serum haptoglobin is low and the LDH high. LDH levels in megaloblastic anaemia can ve very high.

This patients had a low serum B12 and was treated with parental B12 (1mg alternate day for 5 doses) and was evaluated for cause of vitamin B12 deficiency. As Schilling’s test was not available a diagnosis of pernicious anaemia was made by documenting gastric atrophy and anti-parietal cell antibodies.

Calreticulin and Myeloproliferative Neoplasm

Myeloproliferative neoplasm (polycythaemia vera [PV], essential thrombocytosis [ET], progressive myelofibrosis [PMF]) are a group of diseases that are characterised by increased proliferation of blood cells, splenomegaly, myelofibrosis, thrombosis and risk of malignant transformation.  The year 2005 was a landmark year for myeloproliferative diseases. Four groups of scientists identified the presence of JAK2V617F mutations in PV. This mutation is present in about 98% patients with PV. Mutations of exon 12 of the JAK2 gene can be found in 1-2% of the PV. These patients do not show the JAK2V617F mutation. The discovery of these mutations gave a genetic definition PV making diagnosis objective.

PV is diagnosed by the presence primary erythrocytosis in the precession of a JAK2 mutation referred to above. Chronic myeloid leukaemia is diagnosed by demonstrating the BCR-ABL1 translocation. JAK2V617F is also present in 50-60% of ET and PMF. Mutation of the gene MPL is found in 1-2%  patients of ET and 5-10% of the patients with PMF. The presence of these mutation helps make diagnosis. However, The diagnosis of PMF and ET in a large proportion of patients requires exclusion of a reactive disorder and other myeloproliferative diseases because these patients (38-49% of ET and 30-45% of PMF) have no genetic marker.

Two publications have shown that a large proportion of the patients with ET and PMF who do not have JAK have mutation calreticulin (CALR) (N Engl J Med. 2013;369(25):2391-2405,  N Engl J Med. 2013;369(25):2379-2390). In addition to ET and PMF CALR mutations are found in the MDS/MPN overlap disorder and refractory anemia with ring sideroblasts with thrombocytosis (RARS-T). They are rare or absent in other myeloid or lymphoid neoplasms or solid tumors.

Calreticulin (CALR) is a major calcium binding protein. The gene for calreticulin is located on 19p13.2. About a quarter of ET and MF have mutation in the CALR gene. All CALR mutations are localised to exon 9 and generate a 1bp frameshift. As a result of this most or almost all the C terminal negative amino acids and calcium binding sites are lost.  There is a complete loss of the KDEL endoplasmic reticulum binding sequence. These mutations have been identified in the haemopoietic stem cell and progenitor compartments. CALR mutations and JAK2 mutations are mutually exclusive.

CALR mutated myeloproliferative disease have a distinct clinical profile. These patients have a lower haemoglobin, lower leukocyte count, higher platelet count and a lower risk of thrombosis. Patients of PMF carrying a CALR mutation have a longer survival than those carrying JAK2 or MPL mutations. Patients with ET carrying the CALR mutations have a longer survival than those carrying the JALK2 mutation. There is no difference between the survival of ET patients carrying CALR mutations and MPL mutations.

Mutated CALR appears to stimulate STAT pathway. It appears to physically bind with the thrombopoietin receptor to stimulate STAT. The erythropoietin receptor is not needed for this action (Blood. 2015;10.1182/blood-2015-11-681932Blood. 2015;126:LBA-4).




The BCR-ABL1 Gene

CML Pathogenesis-600pxBCR-ABL1 is a fusion gene formed as a result of the t(9;22)(q34;q11) chromosomal translocation, the translocation that results in the formation of the Philadelphia chromosome. The Abelson murine leukaemia viral oncogene homolog 1 (ABL1) gene from 9q34 is translocated downstream to a region at 22q11 known as breakpoint cluster (BCR). The fusion gene encodes for a constitutionally active tyrosine kinase that has been shown to drive the expression chronic myeloid leukaemia phenotype. BCR-ABL1 gene has also been on implicated in the pathogens is of acute lymphoblastic leukaemia and in rare cases of acute myeloid leukaemia. The gene has been targeted with unparalleled success by the first tyrosine kinase inhibitor approved in clinical practice, imatinib.

 Molecular Biology of BCR-ABL

The ABL1 proto-oncogene is located on chromosome 9 at q34. Chromosome 22 has the BCR gene at 22q11. The ABL1 gene translocated downstream to the BCR gene as a result of the t(9;22)(q34;q11) translocation. ABL1-BCR translocation also occurs and may express but is of no clinical significance.


Molecular Biology of CML

The BCR-ABL1 fusion gene and it’s variants

The breakpoint of the ABL1 gene may be upstream exon 1a, between exon 1a and 1b or downstream exon 1b but it is almost always upstream exon 2. With rare exceptions all transcripts of BCR1-ABL1 gene have exon 2-11 of the ABL1 gene. The BCR breakpoints are variable and determine the size as well as the pathogenic properties of the BCR-ABL1 gene.. The breakpoint on the BCR gene are clustered in three regions known major cluster, minor cluster and micro cluster (Table 1). Depending on the location of breakpoint  on the BCR gene three types of protein are synthesized. The p210 transcript is associated with CML and some patients with Philadelphia positive acute lymphoblastic leukaemia. The shorter p190 transcript is associated with philedelphia positive acute lymphoblastic leukaemia and some patients of chronic myeloid leukaemia. The CML that carry this mutation show monocytosis and have a more aggressive course. The p230 is the largest and the rarest of the BCR-ABL1 transcripts. It is associated with a more indolent course and is found in patients with the rare chronic neutrophilic leukaemia. Atypical transcripts e1a3, e13a3 and e6a2 have been described.

Table 1: The BCR-ABL1 fusion genes

 Major Cluster  Minor Cluster  Micro-Cluster
 Synonym M-Cluster m-cluster µ-cluster
Location exons 12-16 Between alternative exon 2, e2’ and e2 between exons e19 and e20
Protein p210 p190 p230
Associated Leukaemia  CML, e14a2 shown to have thrombocytosis in some studies., Ph+ ALL  Ph + ALL; CML that tends to have monocytosis and an agressive course  Chronic Neutrophilic Leukaemia, Small reports describing patients with a course resembling classical CML

Mutagenicity of BCR-ABL1

ABL1 is a nuclear kinase whose activity is tightly regulated by the cell. BCR-ABL1 translocation results loss of regulation and the kinase is  cosntitutively active. Sustitution of ABL1 at the N termnal by segments of the BCR gene result in the synthesis of a protein that has the capacity to dimerise. Dimerisation transphosphorylates and then aurtophsophorylates the the kinase fully activating it. The precise mechanism how BCR-ABL1 leads to chronic myeloid leukaemia is not known but activation of  phosphatidylinositol kinase, RAS/Mitogen activated protein kinase and JAK/STAT pathway has been demonstrated in BCR-ABL1 positive cells. These pathways are involved in cellular growth and differentiation. The BCR-ABL1 kinase also phosphorylates proteins involved in adhesion and migration and this may have a role in premature release of myeloid cells in circulation. CML cells have a two to sixfold increase in reactive oxygen species and have impaired DNA repair. Reactive oxygen species can induce DNA double strand breaks. The results is additional mutations and these are believed to be responsible for blast crisis and acclerates phase.

Tyrosine kinase inhibitors targeting the BCR-ABL1 protein induce a remission in most patients of CML. About half the patients who have achieved sustained complete molecular response relapse on discontinuation of the tyrosine kinase inhibitors. This suggests that the stemat least some CML stem cells are not BCR-ABL1 dependent for growth. Experimental observations support this hypothesis.

Targeting the BCR-ABL1 Gene

The BCR-ABL1 gene was the first gene to be targeted by a tyrosine kinase inhibitor, imatinib. Imatinib was followed by dasatinib, nilotinib, Busotinib and Panotinob. Imatanib, Dasatinib and Nilotinib are approved to first line use. Imatinib has resulted in a 85% 8 year survival. Dasatinib and nolitinib are active in imatinib resistant CML and are now approved for first line use. Drug resistance results from mutations in BCR-ABL1 kinase. The T315I mutation or the gatekeeper mutations impaires access of TKIs to the BCR-ABL1 kinase making most drugs inactive. Panotinib can inhibit the T315I mutation.


Further Reading

Barnes DJ Melo JV. Molecular Basis of Chronic Myleoid Leukaemia. In Chronic Myeloproliferative Disorders: Cytogenetic and Molecular Anomalies. Bain Barbra J (Ed) 2003.

Factor XII

Factor XII is a coagulation factor that initiates the intrinsic (contact) coagulation system. It is for this reason that it is also known as contact factor. It was described in 1955 in a patient John Hageman and hence it is also known as Hageman’s factor. It does not have a role in normal coagulation but may provide a link between inflammation and coagulation. It may have a role in pathological thrombosis.



The Gene for factor XII is situated on chromosome 5 (5q33-qter). The gene is 12 kilobases in length and contains 13 introns and 14 exons. It has an oestrogen responsive element in it’s promoter.


Biochemistry of Factor XII

Factor XII is a 596 amino acid single chain β-globin zymogen with a molecular weight 80kDa. It is primarily produced in the liver. Factor XII is 17% glycosylated. The plasma concentration of factor XII is 40 μg/ml (500 nmol/L) and it has a half-life of 2 to 3 days.


It is the following domains (from the N-terminal to the C terminal)

  1. N-terminal fibronectin domain type II (exons 3,4)
  2. Epidermal-growth-factor like domain (exon 5)
  3. Fibronectin domain type I (exon 6)
  4. Epidermal growth factor like domain (exon 7)
  5. Kringle domain (exon 8 and 9),
  6. Proline rich region
  7. The catalytic domain (exon 10-14) at the COOH end.

Kallikrein splits the bond between Val352 and Arg353 activating factor XII to XIIa (see activation). activation converts factor XII into a two chain structure held together by a disulfide bond between Cys340-Cys367.  The heavy chain is responsible for binding of factor XIIa to anionic surfaces (52 kDa) and a light chain (28 kDa) that has the enzymatic activity.



Factor XII can be activated to XIIa by the following

  1. Contact with negatively charged surfaces: Factor XII is activated by contact with negatively charged surfaces e.g. glass, kaolin, dextran sulphate, ellagic acid and bismuth subgallate. This rection forms the basis of the activated partial thromboplastin test. As none of these activators are encountered under physiological circumstances it is not the mechanism by which factor XII is activated in vivo for normal haemostasis. It has been hypothesized that contact with anionic surfaces provided by polyphosphates results in a conformational change and activation of factor XII. The mechanism of this activation is not clear.
  2. Activation of By Kallikrein: Factor XII, prekallikrein and high molecular weight kininogen can form a complex on anionic phospholipids of membranes. This, as discussed above, leads to a conformational change in factor XII leading to it’s activation. XIIa splits kallikrein from prekallikrein that in turn activates factor XII leading to a mutual activation loop. Activation involves a cleavage of the peptide bond between Arg 353-Val354 that converts a single chain factor XII to a two chain factor XIIa that are connected by a single disulphide bond between Cys340-Cys367.



Actions of Activated Factor XIIa

Factor XIIa activates coagulation by the intrinsic pathway by activating factor XI. The ability of factor XIIa to split prekallikrein to kallikrein links it to inflammation and fibrinolysis. Kallikrein is converts plasminogen to plasmin, pro-u-PA to u-PA and cleaves high-molecular weight kininogen to give bradykinin. Factor XII is pro-inflammatory and promotes thrombolysis by these actions.




The principle inhibitor of factor XIIa is C1-inhibitor. Antithrombin, α1-antitrypsin, α1-antiplasmin and α2-macroglobulin are the other inhibitors of factor XIIa


Role of Factor XII in Haemostasis

Patients with factor XII deficiency do not suffer bleeding following trauma or surgery despite having a prolonged aPTT. The role of factor XII in haemostasis is complex and unclear. There are many divergent pieces of evidence from experimental and epidemiological studies. These include:

  1. Deficiency in factor XII in mice has been shown to protect against thrombosis induced by injury.
  2. Anti-factor XII monoclonal antibody has been shown to reduce fibrin formation in collagen coated tubes perfused by human blood.
  3. The same antibody has been shown to reduce platelet and fibrin deposition in a baboon graft model
  4. Epidemiological studies have shown an inverse relation between factor XII levels from between all cause mortality and factor XII levels for factor XII levels between 10-100% but no increase in patients with factor levels ≤10%.

Factor XII and Disease

Mutations in factor XII have been associated with type III hereditary angioedema (HE) type III. Unlike patients of type I and II HE who have a defect of C1 inhibitor, patients of Type III HE have point mutation in factor XII resulting in substitution of threonine at position 309 with arginine or lysine.



Evaluation of a Bleeding Patinet

From the point of evaluation of a bleeding patients haemostasis can be divided into the following parts.

  1. Platelets,
  2. Vessel wall
  3. Coagulation system: The coagulation system has the three parts intrinsic pathway, extrinsic pathway and common pathway.  The framework of intrinsic pathway, extrinsic pathway and common pathway are useful to understand how to interpret coagulation tests but does not reflect how coagulation proceeds in vivo.
    1. Intrinsic pathway: Coagulation by the intrinsic pathway starts by contact activation of factor XII, this in turn sequentially activates factors XI and IX. Activated factor XI with its cofactor activated factor VIII lyse and activate factor Xa. Factor XII has no role in coagulation in vivo.
    2. Extrinsic pathway: Coagulation by the extrinsic pathway involves activation of factor VII by tissue factor.  Activated factor VII then activates factor X.
    3. Common Pathway: Common pathway includes factor X, its cofactor factor V, prothrombin and thrombin
  4. Thrombolytic system.

The first step in the evaluation of a patients with a bleeding tendency is to perform screening tests to determine which part of the clotting system  is involved. This can be done with the use of the following tests.

  1. Prothrombin time: Prothrombin time asses the efficiency of extrinsic coagulation pathway.
  2. Activated Partial Thromboplastin Time: Activate partial Thromboplastin time assess the efficiency of intrinsic coagulation pathway
  3. Thrombin Time: Thrombin time asses the amount and quality of fibrin.
  4. Platelet Counts

The results of first-line tests are interpreted as follows.

    1. All tests deranged (Prolonged PT, APTT and TT and Low platelets): Derangement of all screening tests for coagulation is seen in patients with disseminated intravascular coagulation, coagulopathy of chronic liver disease, and acute live cell failure. Patients who have received a massive transfusion may also show this pattern of first line tests. In addition to there being a history of massive transfusion, these patients do not show increase in fibrin degradation products (D-dimer).
    2. Global coagulation defect with normal platelet counts (prolonged PT, prolonged APTT, prolonged PT normal platelets): All three first-line tests are abnormal in patients with primary fibrinogenolysis, fibrinogen deficiency, dysfibrinogenaemia, liver disease or treatment with conventional heparin.
    3. Global coagulation defect with normal fibrinogen and platelets (Prolonged PT and APTT normal TT and Platelets):
      1. Fibrinogen synthesis is not vitamin K dependent. Lack of vitamin K coenzyme activity, as is seen in vitamin K deficiency or use of warfarin like anticoagulants, affects the factors of the intrinsic system (factor II and IX ) and extrinsic system (factor VII). The PT and APTT prolong but TT which depends only on fibrinogen remains normal.
      2. The components of the common pathway include factor V, X and factor II. Deficiency of these factors or the presence of inhibitors against any of these factors causes prolongation of PT and APTT but a normal TT.  A rare autosomal recessive disorder, combined factor V and VIII deficiency can cause prolongation of PT and APTT with a normal TT.
      3. Some patients with liver disease may have a prolonged PT and TT.
    4. Isolated prolongation of APTT (Normal PT, Prolonged APTT, Normal TT, Normal Platelets counts): The commonest cause of isolated prolongation of APTT is deficiency or inhibitors of factor VIII or factor IX. Deficiency of other components of the intrinsic pathway including factors XI and XII and prekallikrein can cause isolated prolongation of APTT. Of these only deficiency of factor IX deficiency causes bleeding. Deficiency on factor XII and prekallikrein are not associated with bleeding.
    5. Isolated Prolongation of PT (Prolonged PT, Normal APTT, Prolonged TT and normal platelet count):
      1. The commonest cause of isolated prolongation of PT is a deficiency of factor VII. This may be seen in
        1. Vitamin K deficiency/Use of Warfarin type oral anticoagulants: Factors II, VII, IX and X are vitamin K dependent and have half-lives of 65hrs, 5hrs, 25hrs and 40hrs respectively. When vitamin K co-factor activity is not available either as a result of vitamin K deficiency or the use of warfarin type of oral anticoagulants, the synthesis of vitamin K dependent factors decreases.  The half-life of factor VII is considerably shorter than that of other factors and the levels of factor VII fall the earliest. This results in a prolonged PT but a normal APTT. With time other factor levels fall and the APTT also prolongs.
        2. Early Liver disease
      2. The other causes of isolated prolongation of PT include inherited factor VII deficiency, dysfibrinogenaemia or mild factor VIII, IX or IX deficiency
    6. Isolated prolongation of TT: Isolated prolongation of TT is seen in fibrinogen deficiency or dysfibrinogenaemia
    7. Coagulopathy with normal first-line coagulation tests: May be seen in platelet function defects, vascular coagulation defects, mild von Willebrand’s disease, factor XIII deficiency, disorders of fibrinolysis, dysfibrinogenaemia and α2-Plasmin inhibitor deficiency.
    8. Isolated thrombocytopenia: Isolated thrombocytopenia may be seen in bone marrow failure syndromes, immunethrombocytopenia, following viral infection and in patients with hypersplensim or inherited thrombocytopenia.

Thrombin Time

Thrombin time is the clotting time of plasma on addition of thrombin. It measures the amount and quality of fibrinogen. Thrombin time is prolonged when the fibrinogen levels fall below 70-100mg/dL. The levels of other coagulations factors do not affect thrombin time. Thrombin time is also prolonged by the presence of fibrin/fibrinogen degradation products. It can also be prolonged with a very high fibrinogen levels. Dilution of test plasma normalizes the thrombin time in such patients.


The tests is performed on platelet poor plasma. Thrombin solution is added to PPP and the time for the clot to form is measured. The nature of the clot is also observed. The clot may be transparent or opaque. The test is performed with commercially available bovine thrombin. The concentration of thrombin is adjested to get a thrombin time of 15 on normal plasma. If a more concentrated thrombin is used mild defects in fibrinogen may not be detected. The test is performed with paired test as well as control samples. The thrombin time is the mean of the two readings. Normal thrombin time is two ±2 seconds from the control.

Causes of Prolongation

  1. Therapy with unfractionated heparin. Low molecular weight heparin does not alter the thrombin time appreciably.
  2. Hypofibrinogenaemia and occasionally hyperfibrinogenaemia may prolong thrombin time. The mechanism of prolongation by hyperfibrinogenaemia is unclear but hyperfibrinogenaemia may interfere with fibrin polymer formation.
  3. Dysfibrinogenaemia
  4. Paraproteins may interfere with polymerization of fibrin and prolong thrombin time.

Short Thrombin Time

The thrombin time is shortened when the coagulation is activated.

Nature of the Clot

Transparent fluffy clots indacetes an anomaly in fibrin polymerization. This seen with dysfibrinogenaemias either congenital on seen in liver disease.

Reptilase Clotting Time

Reptilase clotting time is a variation of thrombin time. Clotting is induced by reptilase, an enzyme prepared from snake venom. This test is unaffected by heparin. In samples contaminated with heparin the thrombin time is prolonged where as the reptilase time is normal. On the other hand dysfbrinogenaemia affects the reptilase clotting time more than thrombin time. In patinets with hypofibrinogenaemia the prolongation is equal.