E-Cadherin on Cancer malignancy

E-Cadherin on Cancer malignancy

introduction
The majority of cancers (>90%) are classified as carcinomas originating in the epithelial cells lining the exterior surfaces and internal cavities of the body. These cells exhibit unique morphological characteristics including extensive intercellular complexes, intercellular and cell-cell adhesion to a stable matrix, and distinct apical and basolateral portions of the plasma membrane.

The nature of well-differentiated epithelium allows individual cells to function collectively as a single organized unit Changes in the level of adhesion molecules between cells are important for the development of aggressive carcinomas, especially changes in E-cadherin expression.2 E-cadherin is an adherens junction protein, which through homotypic interactions maintains the barrier function of the epithelium.3 An important hallmark of cancer is the ability of cancer cells to break free from the primary tumor and migrate to distant sites in the body to form new tumors. This metastatic ability generally arises because of the progressive loss of the epithelial characteristics of cancer cells. Currently, E-cadherin is a very active research theme in the field of tumorgenesis.1,2

E-Cadherin Gene and Protein Structure
E-cadherin is a member of the classic cadherin proteins found in epithelial tissue. This protein is encoded by the CDH1 gene. Synonyms of the CDH1 gene include Arc-1, CD324, CDHE, E-cadherin, ECAD, LCAM, Uvomorulin. CDH1 is located on chromosome 16, the long arm of q22.1. This gene is 120 kDa in size, with 16 exons separated by 15 introns. The exons cover an area of ​​up to ~100 kb with base sequences ranging from 115-2245 bp (base pair). Introns range from 120 bp (intron 4) to 65 kb (intron 2). The intron-exon boundary is well preserved compared to other classical cadherins, and in intron 1 a 5′ high-density CpG island was identified that has a role in transcriptional regulation. This island covers the exon 1 to exon 2 area of ​​the human E-cadherin gene, while other exons do not have such features, including the largest exon, exon 16 with 2245 bp (figure 1).2,3

Figure 1. Genomic structure of the human E-cadherin gene

Figure 1. Genomic structure of the human E-cadherin gene. The exon positions are indicated in the color box by the number of base pairs (bp=base pairs) of each exon. The connecting lines are introns. The area from exon 1 to exon 2 on the order of about 1500 bp is a part of a high-density CpG island. (quoted from literature no. 2)

E-cadherin is a type-I transmembrane glycoprotein that is localized to the adherens junction and basolateral membrane of epithelial cells. E-cadherin has several names, including cell adhesion molecule (CAM) 120/80, CD324 antigen, Epithelial cadherin, cadherin 1, cadherin 1 precursor, cadherin type 1, calcium-dependent adhesion protein. E-cadherin is transported from the Golgi complex to the cell surface as a pro-protein and binds to -catenin early in its biosynthesis, whereas P120-catenin and -catenin (which binds to -catenin) only bind after the complex has reached the cell surface. ,4

The structure of E-cadherin consists of 1 extracellular domain, 1 single transmembrane segment and 1 cytoplasmic domain. The extracellular domain consists of 5 repeating extracellular cadherin domains (EC1-5) which bind calcium ions to form rigid linear molecules. The cytoplasmic domain interacts with the actin cytoskeleton through a linking molecule, a protein complex consisting of -, -, and -catenin. – and -catenin share significant homology and bind to a specific domain at the C-terminal E-cadherin. Alpha-catenin links – or -catenin to the actin cytoskeleton (figure 2).1,2

Figure 2. Schematic illustration of E-cadherin in adherens junction

Figure 2. Schematic illustration of E-cadherin in adherens junction. Terlihat E-cadherin homodimer pada selaput sitoplasma sel yang berdekatan. Wilayah juxtamembrane dengan molekul yang berinteraksi dengannya juga ditampilkan. CM – membran sitoplasma; AJ – adherens junction; ED – domain ekstraseluler; ID – domain intraseluler; AC – sitoskeleton aktin; 1-beta-catenin; 2-alpha-catenin; 3-P120. (dikutip dari kepustakaan no. 2) 

Alpha-catenin is structurally similar to vinculin, a major component of the fibroblast membrane of microfilaments, beta-catenin exhibits homology with Armadillo from Drosophila melanogaster and gamma-catenin is identical to placoglobin, a protein found in desmosomes.

The C-terminal cytoplasmic domain with ~150 residues is highly sequenced, and has been experimentally shown to regulate the intercellular binding function of the E-cadherin extracellular domain through interactions with the cytoskeleton. The juxtamembrane region of the cytoplasmic end of cadherin has been identified as a functionally active area that determines cadherin clustering and adhesion strength; one of the proteins involved in cell clustering and adhesion is p120ctn.2 Binding of the cytoplasmic domain of E-cadherin with -catenin and p120-catenin will stabilize E-cadherin molecules on the cell surface.5

The N-terminal structure of the extracellular domain of E-cadherin has the most adhesive activity. This part of the molecule also has bonding regions for calcium ions located between the chains. Intercellular adhesion is mediated by homophilic interactions of the extracellular domain of E-cadherin via a lateral dimerization process. Parallel dimers can bind to dimers from adjacent cells by forming adhesion points. These findings indicate the presence of bonds between cadherins on the cell surface

E-Cadherin Functions
E-cadherin is one of the most important molecules in intercellular adhesion in epithelial tissue.2 Adhesion between adjacent cells occurs through homophilic interactions between E-cadherin molecules. The first interaction occurs between adjacent cells through trans-interaction, then the same cell forms a zipper-like structure through lateral association/unification (cis-interaction).

The adhesion function of E-cadherin plays an important role in epithelial physiology. The complete form of the E-cadherin/catenin complex together with associated actin filaments forms an adherens junction, which fuses 2 plasma membranes with an intercellular gap of only ~25 nmb. This very narrow association stops the movement of individual epithelial cells (known as contact inhibition for motility) and allows organizing these cells into tightly bound layers with functionally distinct apical and basolateral plasma membrane domains. E-cadherins mediating adherens junctions are also important for the formation of tight junctions, an important epithelial structure that forms an apical permeability barrier that blocks the passive diffusion of solutes between the lumen and the interstitial space.

Figure 3. E-cadherin at Tight Junction and Adheren Junction (quoted from literature no. 6)

Figure 3. E-cadherin at Tight Junction and Adheren Junction (quoted from literature no. 6)

In addition to immobilizing epithelial cells in their respective tissue layers, E-cadherin can also inhibit growth factor (GF) mediated proliferation signaling (ie contact growth inhibition), thereby maintaining integrity and maintaining tissue function. Furthermore, E-cadherin can influence various processes that occur during embryonic development, such as cell sorting and epithelial to mesenchymal transition (EMT), and even maintain embryonic stem cell pluripotency.

E-cadherin expression in embryonic development occurs very early, starting at the two-cell stage. Epithelial differentiation and polarization (basic processes for cell differentiation) occur early on ontogeny in the morula stage, when the embryo condenses and each cell undergoes polarization along the apicosal axis to produce an epithelium-like phenotype. E-cadherin plays an important role in blastomere adhesion and early embryonic capacity to condense. E-cadherin is expressed in the membrane even before morula compaction occurs, is distributed in a non-polar manner and has not shown an adhesive function. The mechanism that makes E-cadherin functional is not known with certainty, but it clearly involves the process of protein phosphorylation.2,3

Another important E-cadherin function under development is controlling mesenchymal epithelial conversion. In contrast to well-differentiated epithelium, mesenchymal cells exhibit an elongated morphology with asymmetric edges, forming only transient adhesions with surrounding cells and the extracellular matrix, also producing various enzymes that can degrade the matrix.1

E-cadherin is mainly expressed on the surface of epithelial cells and is responsible for the formation and maintenance of tissue architecture, maintaining stable intercellular relationships and normal epithelial cell polarity. In addition, E-cadherin is important in regulating the development, integrity and organization of epithelial cells
Intercellular interactions mediated by the E-cadherin/catenin complex play a major role in regulating motility, proliferation, cell differentiation and inhibiting cell proliferation when cell-to-cell contact occurs. The loss of cell-to-cell contact will allow beta-catenin to translocate into the nucleus and stimulate cell proliferation. Reconnected E-cadherin will cause beta-catenin to return to the membrane so that proliferation decreases. This event is known as “contact inhibition”. Loss of inhibitory contact due to E-cadherin mutations, or other causes, is a key characteristic of carcinoma

Therefore, in order to maintain the dynamics of a single layer of epithelial cells, E-cadherin is rapidly removed from the plasma membrane, recycled and returned to the cell surface to then form new connections. The degree of adhesion strength formed can be modulated by regulation of transcription and/or protein degradation through the ubiquitin-proteosome pathway. In addition, junction protein recycling is an alternative mechanism that allows cells to undergo rapid morphology in response to extracellular stimuli, such as low levels of Ca2+ and growth factors such as hepatocyte growth factor (HGF).

Until recently, cadherin was thought to be involved only in homophilic interactions, but now E-cadherin has also been shown to be a ligand for two integrins, alphaEbeta7 and alpha2beta1. The first interaction serves to maintain intraepithelial lymphocytes in the mucosal tissue, while the second contributes to the organization of the epithelial layers

The Role of E-cadherin in Cancer Cells

a. E-cadherin expression in cancer
The progressive accumulation of somatic mutations in a number of different genes characterizes the process of tumorigenesis (tumor formation). Many genes are involved in the process of tumorigenesis and are components of one of the many signal transduction pathways of molecular networks. It is now clear that epithelial malignancy in some aspects can be explained by changes in the adhesion properties of neoplastic cells
In stark contrast to their parent epithelial cells, carcinoma cells usually do not show contact inhibition of either their motility or growth. The most prominent feature shown by E-cadherin is that it promotes contact inhibition so that E-cadherin is a strong molecular barrier that must be overcome if tumor cells are to proliferate, detach, and spread. Loss of function of E-cadherin is often a marker of molecular changes that occur during tumor progression. This is due to a variety of mechanisms, including transcriptional repression, epigenetic silencing, inactivating mutations, endocytosis and fragment separation by proteolysis.

In addition to its role in normal cells, this gene can play a major role in the transformation of malignant cells, especially in tumor development and progression. Emphasis of E-cadherin expression is considered ap as one of the main molecular events responsible for the occurrence of intercellular adhesion dysfunction. Most tumors have an abnormal cellular architecture, and hence, loss of tissue integrity can lead to local invasion. Thus, loss of function of E-cadherin as a tumor suppressor protein correlates with increased tumor invasiveness and metastasis, so E-cadherin is often referred to as a suppressor of invasion gene.2 Ultimately, reduced or absent E-cadherin expression is an indication of outcome. poor clinical condition in several malignancies such as esophageal tumors, lung tumors, cervical tumors and Willm’s tumors.1,5

The metastatic ability of cancer cells is also thought to arise due to the progressive loss of epithelial characteristics of cancer cells by adopting more mesenchymal phenotypes in a process called EMT. Downregulation of E-cadherin is a standard feature in the development of EMT and loss of E-cadherin function in cancer plays an important role in the transition to a malignant phenotype.

E-cadherin-mediated loss of epithelial cell-to-cell binding is a hallmark of the transformation of benign lesions to invasive metastatic cancer. However, there is evidence that E-cadherin also plays a role in signal transduction pathways including wnt signaling, along with other important molecules involved, such as beta-catenin.

Expression of E-cadherin can suppress Wnt/b-catenin signaling by binding to -catenin at the site of cell-to-cell contact. In the absence of Wnt ligand binding to the Frizzled receptor, -catenin will be phosphorylated by CK1 kinase and GSK-3B, which in turn undergoes ubiquitination and proteasomal degradation.

However, once Wnt signaling is initiated due to lack or absence of E-cadherin expression, phosphorylation of -catenin will be inhibited, leading to accumulation of free -catenin in the cytoplasm and subsequently translocated into the nucleus to form an active complex of transcription factor -catenin/TCF/ LEF (T cell factor-lymphoid enhancer factor) and ultimately results in the expression of Wnt target genes. One of the target genes of this complex is encoding the c-MYC protein, which explains why constitutive activation of the Wnt pathway can lead to malignancy (figure 4).

Gambar 4. Wnt signaling (dikutip dari kepustakaan no. 3)

Gambar 4. Wnt signaling (dikutip dari kepustakaan no. 3)

b. E-cadherin Fragment Cleavage in cancer
Strong intercellular binding is a major barrier to cancer cell mobility, and the loss of cell-cell adhesion by E-cadherin is a fundamental change that occurs during the development of invasive cancer. Proteolytic processes and the release of membrane protein fragments (called shedding ectodomains) are common pathways by which cells modify the functional properties of membrane proteins. It is now known that proteolysis of E-cadherin fragments can promote tumor growth, survival and motility suggesting that fragmented E-cadherin fragments can convert these tumor suppressors into oncogenic factors.

Cleavage of E-cadherin by -secretase occurs on the extracellular surface of the plasma membrane catalyzed by several proteases, including matrix metalloproteinases (MMP-2, MMP-7, MMP-9, and MT1-MMP), A-disintegrin-and-metalloproteinase ( ADAM10 and ADAM15), plasmin. This cleavage transforms the characteristic 120 kDa mature E-cadherin into an 80 kDa extracellular N-terminal phagment and a 38-kDa intracellular C-terminal fragment. Ectodomain fragments [termed soluble E-cadherin (sE-cad)] are released from the plasma membrane and diffuse into the extracellular environment and even the bloodstream to act as paracrine/autocrine signaling molecules. In contrast, the intracellular C-terminal fragment (E-cad/CTF1) remains bound to the plasma membrane until cleavage occurs at the intracellular surface via -secretase (Presenilin-1/2).

This enzyme breaks the adherens junction and releases a 33-kDa intracellular fragment (E-cad/CTF2) into the cytosol where it plays a role in intracellular signaling. This fragment can also undergo further proteolysis by caspase-3 to produce a 29-kDa fragment (E-cad/CTF3) whose function is unknown (Figure 5).

Figure 5. Cleavage of full-length E-cadherin by -secretase, -secretase, and caspase-3 (cited from literature no. 1)

Figure 5. Cleavage of full-length E-cadherin by -secretase, -secretase, and caspase-3 (cited from literature no. 1)

sE-cad increases motility and invasion
Separation of the ectodomain fragment of E-cadherin was first found in media from breast carcinoma cells. In several subsequent studies, investigators observed a significant increase in sE-cad in the serum of cancer patients from various tumor types and reported that a significant increase in sE-cad was associated with invasive disease course and/or poor prognosis. The first function related to sE-cad is impaired intercellular adhesion based on the observation that in vitro cell therapy with sE-cad decreases cell aggregation and increases migration and invasion. The sE-cad mechanism for these changes involves at least 4 steps: (Figure 5)

1. The cleavage of E-cadherin degrades the whole molecule, so that the reserves of E-cadherin that are competent for the adhesion process on the plasma membrane will be reduced.
2. Because sE-cad has the ability to form homophilic bonds, the full-length E-cadherin homodimer between adjacent cells can be blocked.
3. sE-cad has chemotactic properties and can be trapped in the extracellular matrix and acts as an anchor/anchor of intact E-cadherin molecules from migrating cells.
4. sE-cad causes upregulation (upregulation) of major MMPs (MMP-2, MMP-9, and MT1-MMP) which degrades the basement membrane resulting in tumor invasion into the stroma. Then, because MMP-9 is also known to cleave E-cadherin, protease induction by sE-cad also gives positive feedback to continuously produce sE-cad.

Figure 6. Oncogenic function of sE-cad and E-cad

Figure 6. Oncogenic function of sE-cad and E-cad

sE-cad induces EGFR-dependent growth and survival signaling
It is known that E-cadherin regulates cell signaling processes. Activation of the receptor tyrosine kinase (RTK) occurs via receptor dimerization and autophosphorylation, and clustering of E-cadherin molecules after intercellular homophilic interactions can trigger ligand-independent dimerization and EGFR activation. Since the extracellular domain of E-cadherin can interact with EGFR, cleavage and separation of this domain into the extracellular environment can convert this molecule into a soluble growth factor.
This new function of sE-cad was first demonstrated in breast cancer cells. The EGFR (ErbB) family of RTK consists of 4 members: EGFR (HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These receptors can be both homodimeric and heterodimeric in response to ligand binding. Najy and colleagues, observed that sE-cad can bind to HER2 and HER3, stabilize their interactions, and activate signals to promote proliferation and migration (Figure 6). Because HER2 overexpression is common in breast cancer and is associated with a poor prognosis, proteolytic release of sE-cad may stimulate this pathway to promote aggressive growth and tumor metastasis.
The signaling process by sE-cad can also influence apoptosis through ligand-independent EGFR activation. Studies by David et al., collectively demonstrated that sE-cad acts as an anti-apoptotic factor, which means that sE-cad may play an important role in the survival and initiation of preneoplastic conditions in epithelial tissues.
The relationship between sE-cad and EGFR also suggests a reciprocal relationship in which EGFR activation promotes MMP and ADAM-dependent sE-cad. This suggests a feedback mechanism by which EGFR activation generates sE-cad, which then further activates EGFR signaling to continuously promote oncogenic proliferation and invasion (Figure 6).

E-cadherin cleavage activates Wnt/β-catenin . signaling pathway
The linking component of the cadherin-catenin complex is -catenin, which functions as a major mediator of the Wnt signaling pathway. In the classical Wnt pathway, Wnt ligand binding triggers the accumulation of -catenin, to translocate into the nucleus and bind to the transcription factor T-cell factor/lymphocyte enhancer factor-1 to activate growth-promoting target genes. By forming a complex with -catenin, E-cadherin will effectively bind this protein to the plasma membrane and prevent its transcriptional activity. However, cleavage of E-cadherin destroys this complex and releases -catenin to potentiate the oncogenic Wnt signaling pathway (Fig. 6). This pathway is also directly enhanced by intracellular E-cad/CTF2 fragments. In addition to liberating b-catenin, cleavage of E-cadherin also liberates P120 catenin, which translocates into the nucleus and binds to the Kaiso transcriptional repressor to abolish repression of various target genes of the Wnt/b-catenin pathway. It is also important that the E-cad/CTF2 fragment can remain bound to P120 catenin to enhance its inhibitory effect on Kaiso-mediated transcriptional repression (Fig. 6).
These studies demonstrate a dual mechanism of E-cadherin cleavage that facilitates the signaling of the Wnt/b-catenin pathway. The resulting aberrant activation of the Wnt/b-catenin pathway which functions to support the rapid proliferation of tumor cells and also increases tumor cell survival, as the expression of nuclear E-cad/CTF2 also exhibits suppressive activity against the induction of apoptosis. In addition, E-cadherin cleavage may also promote the acquisition of cancer stem cell phenotypes, because the Wnt/b-catenin signaling pathway is involved in stem cell regeneration. Furthermore, because MMP-7 is a Wnt/b-catenin target that has been shown to cleave full-length E-cadherin, activation of this gene can establish a positive feedback loop for continuous E-cadherin cleavage to ensure continuous oncogenic signaling.

CONCLUSION
1. E-cadherin is an important tumor-inhibiting gene.
2. The involvement of E-cadherin in the wnt signaling process, indicates that the same molecule can have different functions and that E-cadherin can regulate cellular responses shaped by external signals received by the cell. In this case, E-cadherin can regulate the process of migration, proliferation, apoptosis and cell differentiation.
3. E-cadherin cleavage produces protein fragments that are oncogenic, another mechanism that can increase motility, invasion, survival and tumor growth, also plays a role in signaling the WNT pathway to ensure continuous oncogenic signaling.
4. One important future approach in gene therapy is to develop methods to prevent the decrease in E-cadherin levels in tumors. To mencPreventing the potential for metastasis in almost all types of epithelial tumors can be initiated by targeting this molecule. Even so, this will be difficult because the process of decreasing E-cadherin levels is influenced by many different mechanisms, from large mutations and deletions to repression of gene transcription, as well as stimulation of signal transduction from formation of E-cadherin adhesion complexes.
5. Low expression of E-cadherin in tumor tissue or high levels of its fragments (sEcad and E-cad/CTF2) in the circulation signify invasiveness and severe proliferation of tumor cells which will result in poorly differentiated tumors.
6. The levels of E-chaderin and the levels of its fragments (sEcad and E-cad/CTF2) can be used to determine the severity of the disease and the degree of tumor metastasis.
7. E-cadherin and its fragments can determine the prognosis of the disease and the type of therapy in malignancy.

Bibiliography

  1. David JM, Rajasekaran AK, Dishonorable Discharge: The Oncogenic Roles of Cleaved E-Cadherin Fragments. American Association for Cancer Research. 2012.
  2. Slaus NP. Tumor suppressor gene E-cadherin and its role in normal and malignant cells. Cancer Cell International.
  3. Bossche JV, Malissen B. Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs. The American Society of Hematology. 2012.
  4. E-Cadherin (Calsium dependent adhesion molecules).

http://en.wikipedia.org/wiki/cadherin

  1. Alami J, Williams BR, Yeger H. Differential expression of E-cadherin and b catenin in primary and metastatic Wilms’s tumours. BMJ. 2003.
  2. Tight Junction. https://en.wikipedia.org/wiki/Tight_junction Structure

      By :

dr. Suhardi.Mkes.SpA,Dr.dr. Nadirah R. Ridha, MKes., Sp.A(K),Prof. Dr. dr. H. Dasril Daud, Sp.A(K)

BAGIAN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN UNIVERSITAS HASANUDDIN

MAKASSAR

2015

Posted in Cancer | Tagged , , , , , , | Leave a comment

TRANSFUSION IN PREMIUM BABY part two

mechanism of platelet function

mechanism of platelet function

The use of platelet transfusion for neonates with thrombocytopenia and active bleeding is considered appropriate, but there is uncertainty and variation in practice in the wider use of platelet transfusions for prophylaxis in the absence of bleeding. In a prospective multicenter observational study of 169 neonates with a platelet count less than 60 × 109/l, most of the transfusions were prophylactic and administered to the neonate and many were given after the period when major bleeding, including IVH, occurred most frequently. Many infants receive platelet transfusions in the pre-transfusion platelet count range between 25 and 50 x 109/l. In the absence of results from RCTs in this patient group, the recommendation for prophylactic platelet transfusion was made based on clinical experience. 9

Platelets are generally given in doses of 5–10 ml per kg, which should be expected to increase the full-term infant’s platelet count by 50–100 x 109/l.8
Among all the potential risks associated with platelet transfusion, transfusion-associated acute lung injury (TRALI) deserves special consideration because (given the frequency with which neonates experience respiratory distress in the NICU).

Platelet concentrate age-mismatch transfusion

Platelet concentrate age-mismatch transfusion

and possible side effects. Currently, platelet concentrates are collected from adult donors. Adult platelets have distinct phenotypes that differ from the physiological properties of neonatal platelets. Possible harmful effects should be considered and investigated in more detail. TRALI, transfusion-associated acute lung injury; ulVWF, unusually largeVWF.

Neonatal Thrombocytopenia Management Algorithm

Neonatal Thrombocytopenia Management Algorithm

Plasma transfusion
The main indication for plasma transfusion in neonates is correction of bleeding due to deficiency of some acquired coagulation factors. Where possible, the decision to transfuse plasma should be guided by the clinical situation and by appropriate laboratory testing. The use of plasma is not recommended when the primary purpose of the transfusion is to treat hypovolemia. In addition, plasma transfusions should be avoided when safer products can be used to achieve the same therapeutic purpose. For example, virally inactivated recombinant factor concentrates are preferred for the isolated treatment of coagulant factor deficiency. Plasma is usually given at a dose of 10 to 15 ml per kg. This dose can be expected to increase factor activity by 20% in infants without continued consumption of coagulation factors. 8

Table 4. Coagulation factor values in neonates

Table 4. Coagulation factor values in neonates

FFP There is considerable uncertainty about the appropriate use of FFP in neonates, reflecting the lack of evidence in this area. National audits have shown a high proportion of FFP transfusions given for prophylaxis. The prophylactic use of FFP, including preoperatively, is of unproven benefit and the uncertainty is compounded by the difficulty in defining a significant coagulopathy in this age group. 1

Table of interventions and recommended for coagulopathic disorders

Table of interventions and recommended for coagulopathic disorders

Neonates have a different balance of procoagulant and anticoagulant proteins compared to older children but overall hemostasis can be functionally adequate when determined by global measures of hemostasis. This results in different postpartum and gestational coagulation ranges in the first months of life, especially for activated partial thromboplastin time (APTT). Most laboratories rely on previously published neonatal ranges because of the difficulty in obtaining locally derived ranges in this age group but variations in reagents and analyzes can make interpretation of results difficult.

Polycythaemia with elevated Hct may further contribute to prolongation of coagulation time, in particular prothrombin time (PT). In older children and adults, coagulopathy is often defined as a PT or APTT greater than 1.5 times the midpoint of the normal range, but these are more difficult to apply to neonates, especially in very premature neonates given that the ranges may not be definite and broad. In addition, disseminated intravascular coagulation (DIC) is a poorly defined entity in the neonate

Table 5. indications for plasma transfusion

Table 5. indications for plasma transfusion

Routine coagulation screening of premature infants admitted to the NICU may lead to increased transfusions. Coagulation studies should only be performed for selected neonates with evidence of bleeding or a high risk of DIC, such as those with NEC or severe sepsis. Although most neonatal coagulopathy will be secondary to an acquired bleeding disorder, undiagnosed congenital bleeding disorders should also be considered.
Transfusion complications

The safest blood transfusion is one that is not performed. When a transfusion is needed, it is important to be aware of the potential for acute and delayed side effects. Adverse consequences can be minimized through early recognition and prompt therapeutic intervention.10

Noninfectious complications can be acute (occurring within hours of transfusion) or delayed. Acute reactions can be further categorized according to their pathogenesis: immunological versus nonimmunological. 10

Acute hemolytic reactions are the second most common cause of transfusion-related death. Prior to the first transfusion, neonates should be screened for passively transferred RBC antibodies, including ABO antibodies. Infants are at increased risk of passive immune hemolysis from transfusion of ABO-incompatible plasma. Although small amounts of ABO-incompatible plasma (eg, 5 mL/kg) are usually well tolerated. 10

In neonates, acute hemolytic events can be characterized by increased plasma free hemoglobin, hemoglobinuria, increased potassium concentration, and decreased pH. Results from the direct antiglobulin (Coombs) test may confirm the presence of antibodies on the RBC surface. 10

Allergic reactions are rare in neonates. They occur when a patient has formed immunoglobulin (Ig) E antibodies to the allergen in donor plasma. Residual cytokines or chemokines (eg, RANTES) released by stored platelets may also contribute to allergic reactions. Most reactions respond to antihistamines. Severe anaphylactic reactions are rare; some are associated with antiIgA antibodies. 10

Transfusion-associated acute lung injury (TRALI) is the most common cause of transfusion-related death but often remains unrecognized. The recommended diagnostic criteria for TRALI are acute onset of hypoxaemia with bilateral infiltrates on chest radiograph within 6 hours of blood transfusion and no evidence of excessive circulation. 10

Figure 9. Possible pathogenic chain of transfusion lesion components

Figure 9. Possible pathogenic chain of transfusion lesion components

Figure 9. Possible pathogenic chains of components of a transfusion lesion – red cells, microvesicles and lipids – in the context of TRALI. Leakage of blood components into the alveolar space, secondary to inf

lammation and damage to the vascular endothelium, may implicate TRALI.
Premature neonates are at increased risk of fluid overload from transfusion because of the volume of blood components. Metabolic complications are encountered mainly with large transfusions (15 to 20 mL/kg). 10

Nonimmune hemolysis of blood components can occur from overheating, use of intravenous solutions other than normal saline and exposure to hypo-osmotic conditions, bacterial contamination, combined irradiation with prolonged storage, and mechanical damage from rapid infusion through small needles. 10

Conclusion
1. Blood transfusion is a substitution therapy in certain circumstances.
2. Indications for blood transfusion in premature infants are based on hemoglobin levels, clinical conditions and use of assistive devices, if there is nothing if the Hb level is 7 g/dl.
3. Giving blood transfusions must be careful because there are negative consequences.

Bibliography

  1. New H V., Berryman J, Bolton-Maggs PHB, et al. Guidelines on transfusion for fetuses, neonates and older children. Br J Haematol. 2016;175(5):784-828. doi:10.1111/bjh.14233
  2. Howarth C, Banerjee J, Aladangady N. Red Blood Cell Transfusion in Preterm Infants : Current Evidence and Controversies. 2018:7-16. doi:10.1159/000486584
  3. Lisa A. Hensch, Alexander J. Indrikovs, Karen E. Shattuck. Transfusion in Extremely Low-Birth-Weight Premature Neonates: Current Practice Trends, Risks, and Early Interventions to Decrease the Need for Transfusion. Neo Rev. 2015;16(5):287-296. doi:10.1542/neo.16-5-e287
  4. Ohls RK. Transfusions in the Preterm Infant. Neoreviews. 2007;8(9):e377-e386. doi:10.1542/neo.8-9-e377
  5. Conti VS, Azzopardi E, Parascandalo R, Soler P, Montalto SA. Overview of the blood transfusion policy in preterms on the neonatal intensive care unit. Malta Med J. 2013;25(3):46-50.
  6. Chen HL, Tseng HI, Lu CC, Yang SN, Fan HC, Yang RC. Effect of Blood Transfusions on the Outcome of Very Low Body Weight Preterm Infants under Two Different Transfusion Criteria. Pediatr Neonatol. 2009;50(3):110-116. doi:10.1016/S1875-9572(09)60045-0
  7. Unkar ZA, Bilgen H, Yaman A, et al. Effect of multiple transfusions on lipid peroxidation in preterm infants. Clin Chem. 2015;1)(3):S183. http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed14&AN=72265426%5Cnhttp://openurl.ac.uk/athens:_edu//lfp/LinkFinderPlus/Display?sid=OVID:Embase&id=pmid:&id=&issn=0009-9147&isbn=&volume=61&issue=10+SUPPL.+1&spage=S183&pages=S183&date=2.
  8. Brown BYA. Blood transfusion in. 1916;30(4):716-723.
  9. Sparger K, Deschmann E, Sola-Visner M. Platelet Transfusions in the Neonatal Intensive Care Unit. Clin Perinatol. 2015;42(3):613-623. doi:10.1016/j.clp.2015.04.009
  10. Galel SA, Fontaine MJ. Hazards of Neonatal Blood Transfusion. Neoreviews. 2006;7(2):e69-e75. doi:10.1542/neo.7-2-e69
  11. Saunders RA, Purohit D, Hulsey TC, et al. The effects of blood transfusion protocol on retinopathy of prematurity [3] (multiple letters). Pediatrics. 2000;106(2 I):378-380. doi:10.1542/peds.106.2.378
  12. Girelli G, Antoncecchi S, Casadei AM, et al. Recommendations for transfusion therapy in neonatology. Blood Transfus. 2015;13(3):484-497. doi:10.2450/2015.0113-15
  13. R N. “To Transfuse or Not to Transfuse”-A Neonatologist’s Daily Dilemma. J Hematol Thromboembolic Dis. 2016;4(4):1-3. doi:10.4172/2329-8790.1000250
  14. Wylie BJ, D’Alton ME. Fetomaternal hemorrhage. Obstet Gynecol. 2010;115(5):1039-1051. doi:10.1097/AOG.0b013e3181da7929
  15. R. A. Shanmugha Priya, R. Krishnamoorthy, Vinod Kumar Panicker, Binu Ninan. Transfusion support in preterm neonates <1500 g and/or <32 weeks in a tertiary care center: A descriptive study. Asian J Transfus Sci. 12(1): 34-41

By :

dr. Rahmi Utami. SpA.,DR.dr.Nadirah Rasyid Ridha.Mkes.SpA.,Prof. DR.dr.Dasril Daud.SpA

DIVISI HEMATOLOGI-ONKOLOGI

DEPARTEMEN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN UNIVERSITAS HASANUDDIN/

 RS dr. WAHIDIN SUDIROHUSODO

MAKASSAR

Posted in ANEMIA | Tagged , , , | Leave a comment

TRANSFUSION IN PREMIUM INFANT part 1

TRANSFUSION IN PREMIUM INFANT

preliminary
Appropriate transfusion in pediatric patients, especially in premature infants is very important to balance the benefits of transfusion against the risks. These transfusion risks include unpredictable acute transfusion reactions. Additional safety measures are in place for neonatal patients who are particularly vulnerable recipients due to their small size and immaturity of the organs.1

Very low birth weight (ELBW) babies, who weigh less than 1,000 g, about 90% of these babies will require at least one transfusion. The need for transfusion in this population has a number of causes, the most significant of which is anemia due to prematurity. Anemia of prematurity is a multifactorial effect of low body weight in the first few weeks after birth
India ranks first with 23.6% of premature births in the world. Premature neonates are the patient group requiring the most transfusions and approximately 85% of very low birth weight (ELBW) newborns receive transfusions at the end of the hospital stay. The reasons for increased transfusion in premature infants are immature hematopoiesis, poor hematological compensatory mechanisms, blood loss due to frequent laboratory tests, sepsis, necrotizing enterocolitis (NEC), bleeding, and consumptive coagulopathy. (Shanmugha Priya, 2018)

Definition
The standard definition of a neonate is that an infant with the age of newborn up to 28 days after birth and preterm is defined as an infant born alive before 37 weeks of gestation. There are 3 divisions of premature babies according to the time of birth, namely Extremely Preterm (<28 weeks), Very Preterm (28 – <32 weeks) and Moderate – Late Preterm (32 -36 weeks).1,2

Blood transfusion is the process of giving blood or blood-based products from one person to the circulatory system of another. Blood transfusions are associated with medical conditions such as large blood loss due to trauma, surgery, shock and malfunctioning of the red blood cell-forming organs.

Blood transfusion in premature babies
The trigger for transfusion for the neonate will vary depending on the clinical context, including gestational age at birth. General neonatal transfusion guidelines have been developed as a result of neonatal studies especially in very low birth weight (VLBW; <1.5 kg) infants. In neonatal intensive care units (NICUs) most transfusions are given to premature neonates (mostly <32 weeks’ gestation), some of whom will require transfusion after 28 days. In general, infants of all gestational and postnatal ages in the NICU will tend to be transfused using the same guidelines although there is little specific evidence associated with term infants. Thresholds for transfusion are usually based on hemoglobin (Hb) concentration, platelet count and/or coagulation test results.1,2
Overall from the 1990s to the present it has been reported that up to 90% of very low birth weight (ELBW) infants and 58% of premature infants <32 weeks of gestational age receive red blood cell transfusions (PRCs), mainly due to iatrogenic loss and ventilation requirements. .1

Red blood cell transfusion
The majority of very premature neonates (<28 weeks gestation) received at least one red cell transfusion because they were frequently anemic, partly due to loss of blood sampling (note: 0.5 ml blood sample in 500 g (1 ml/kg) infant, approximately equivalent to 70 ml of sample in a 70 kg adult), or sometimes with a larger sample volume than required. The use of cord blood for baseline blood testing for VLBW neonates has been advocated to reduce the need for transfusion. Neonatal transfusions are usually given as additional small volume transfusions to maintain Hb above a certain threshold or because of the presence of surrogate markers of anemia, such as increased episodes of apnea. 4
In general, PRC is beneficial for preterm infants who are ill and in intensive care by increasing circulating hemoglobin (Hb), increasing tissue oxygenation, and reducing cardiac output to maintain the same level of oxygenation. 4

Picture. 1. Fetal circulation scale

Picture. 1. Fetal circulation scale (A) and hemodynamic adaptations after birth (after direct cord clamping [B] or during delayed cord clamping [C]). OD: right atrium; OG: left atrium; DV: right ventricle; VG: left ventricle; AP: pulmonary artery; FO: foramen ovale; AD: ductus arteriosus; PA: arterial pressure; O: organs; yellow: vascular and / or tissue resistance; cross mark: clamping the umbilical cord; arrow direction: blood circulation direction; red arrow: blood is rich in oxygen; blue arrow: oxygen-poor blood; purple: oxygen-poor blood.

The potential benefits of transfusion in this group include increased tissue oxygenation and lower cardiac output to maintain the same level of oxygenation. These benefits need to be weighed against possible adverse outcomes. The major serious complications of prematurity including bronchopulmonary dysplasia, retinopathy of prematurity, intraventricular hemorrhage, and necrotizing enterocolitis can follow neonatal transfusion, all of which cause significant morbidity and mortality in premature infants. 4

Picture. 2. Pathophysiology of anemia

Picture. 2. Pathophysiology of anemia

Picture. 2. Pathophysiology of anemia due to prematurity as described by the interaction of physiological events, occurring late in pregnancy, which is “missed” by prematurely born infants plus premature complications that contribute to reduced red blood cell status and erythropoiesis.

One of the causes of anemia in premature infants is fetomaternal transfusion, which refers to the entry of fetal blood into the mother’s circulation before or during delivery. Antenatal fetomaternal hemorrhage is a pathological condition with a wide spectrum of clinical variations. Although there is no universally accepted definition of the fetal erythrocyte transfer rate that constitutes a fetomaternal transfusion, the range of blood volumes ranges between 10 and 150 mL. Massive fetomaternal transfusion is when a blood volume greater than 80 mL is transferred. Fetal bleeding is secondary to anemia that occurs, it may have adverse consequences for the fetus such as neurologic injury, stillbirth, or death of the newborn. Presentation is often without clear trigger factors. (Wylie, 2010)

. The effect of red blood cell unit preparation and storage on heme, iron, and oxidative status relates to the ability of premature infants to cope with these changes. Red blood cell units are a potential source of heme, iron and free radicals, and these increase with storage age. Hemolysis from red blood cell transfusions can add iron-free hemoglobin to the baby.4

The majority of red cell transfusions for neonates are supplementary small volume transfusions (usually 10-20 ml/kg, usually 15 ml/kg over 4 hours) given to replace losses in the context of premature anemia, especially for VLBW infants. There is very limited evidence to determine the optimal volume for red cell transfusion, particularly with regard to long-term outcomes. Volumes greater than 20 ml/kg may increase the risk of volume overload in patients who are not bleeding. Therefore, in the context of data supporting the restrictive transfusion threshold of patients of all age groups including neonates.5

Blood transfusion policies and methods to ensure their implementation have an impact in reducing the number of red blood cell transfusions. The Hb level is widely used as a marker of the need for transfusion despite its limitations. The specific threshold of Hb at which neonates are transfused varies according to cardiorespiratory status and age after birth, in part following the normal physiologic decline in Hb during the first few weeks of life.

Indications for red blood cell transfusion

Anemia becomes symptomatic when there is an imbalance between oxygen delivery and consumption that may not occur universally at the same Hb for every preterm infant. Symptoms of anemia (eg, desaturation, bradycardia, increased oxygen demand, and tachycardia) are nonspecific and may be due to other causes including sepsis, development of pulmonary conditions (including worsening of respiratory distress syndrome), or gastro-oesophageal reflux. Therefore, PRC transfusion may not result in such clinical improvement. Generally, PRC transfusions are given to keep the Hb level above a certain threshold depending on the level of cardiorespiratory support required. Nearly half of the PRC transfusions given to ELBW infants were administered during the first 2 weeks of life, when there was impaired cardiorespiratory and laboratory blood cell collection; blood loss due to weekly blood sampling during this period averages 10-30% of the total blood volume (10-25 mL/kg). PRC transfusions are also given because of acute blood loss (eg, maternal fetal haemorrhage or placental abruption) or because of clinical symptoms regardless of Hb level, or infants are breathing spontaneously, but with Hb below a certain threshold, with the aim of increasing their body weight1

Algorithm: PRC Transfusion Neonatal Guidelines for

Algorithm: PRC Transfusion Neonatal Guidelines for

Blood Transfusion Threshold
There is no consensus on the threshold at which premature infants should receive PRC transfusions. Hb levels are subjective and vary among different education centers despite recently published national guidelines. The British Committee for Standards in Hematology (BCSH) published revised guidelines in 2016 and a second edition of the American practice guidelines for PRC transfusion was published in 2007. This emphasizes the importance of defining poor cardiopulmonary status. A comparison of guidelines from different countries highlights how varied these thresholds are internationally.1

Blood Transfusion Threshold

Blood Transfusion Threshold

Benefits of Blood Transfusion
PRC transfusions are life saving when given to replace acute bleeding loss by replenishing circulating blood volume. The potential benefits of PRC transfusion for anemic premature infants include prevention of apnea and weight gain.3

Lack of Blood Transfusion
Complications of PRC transfusion include increased plasma free iron and possible iron overloading of the liver with iron in very low birth weight (VLBW) infants, which is a clinical implication of iron being removed from red blood cells during storage of red blood units as a result of oxidative damage to the RBCs. RBC membrane and hemoglobin. 7
Because of the physiological importance of iron and the potential toxic effects of free iron, the body is equipped with a very precise homeostatic mechanism to regulate iron bioavailability. The ability of albumin to bind iron appears to be of great importance as a defense mechanism against iron-induced oxidative damage. Serum albumin levels are lower in premature infants compared to term infants. Serum albumin in premature infants is highly susceptible to oxidative stress. Caeruloplasmin, which converts iron into the form required to bind to transferrin, may also be low in premature infants. Serum hepcidin concentrations are lower in preterm infants than in term infants. 7
Free heme has pro-oxidant and pro-inflammatory activities and many other potentially toxic activities. The potential toxicity of heme is limited by the presence of the heme-binding protein hemopexin. Thus adequate availability of hemopexin is required to prevent the toxic effects of heme. Premature infants have very low levels of hemopexin which makes them susceptible to the effects of excessive heme-mediated transfusion. 7

Figure 3. Hemolysis process

Figure 3. Hemolysis process

Storage of red blood cell units was associated with a gradual increase in malondialdehyde (MDA). MDA is a marker of lipid peroxidation. It is known that oxidative stress, normal aging and aerobic incubation lead to the release of free iron from Hb in erythrocytes. This suggests that lipid peroxidation in the RBC membrane may contribute to the loss of iron and Hb. Thus the evidence suggests that oxidatively mediated hemolytic changes in the RBC membrane and damage to iron-binding proteins lead to the release of iron from red blood cells into the extracellular medium during PRC storage. 7

There is evidence that iron released in erythrocytes can mediate oxidative damage to cell membranes, leading to hemolysis. It can be further oxidized to produce superoxide, methemoglobin and free iron. Methemoglobin is relatively unstable and will easily release the heme portion from the heme bag. Further oxidation of the heme molecule causes the release of free iron. Thus the lack of antioxidant protection contributes to oxidative damage to cell membranes and binds to proteins such as iron as Hb. The potential adverse effects of biochemical storage and the validity of stored erythrocytes are termed “storage lesions”.7

Many of these involve iron-mediated transfusion-induced factors such as infection and oxidative stress, changes in immune function and also other factors such as nitric oxide (NO)-mediated and responsive changes. 7

Figure 4. The process of formation of prooxidation and proinflammatory in hemolysis

Figure 4. The process of formation of prooxidation and proinflammatory in hemolysis

Perhaps the greatest controversy surrounding PRC transfusion in premature infants is its association with necrotizing enterocolitis (NEC). There are reports of a transient association between PRC transfusion and NEC occurring within 72 hours of this transfusion. 25–35% of NEC cases are transiently associated with RBCT and the terms “transfusion-associated NEC” or “transfusion-associated acute bowel injury”. 1, 7

Figure 5. The process of NEC in PRC . transfusion

Figure 5. The process of NEC in PRC . transfusion

Increased superior mesenteric artery blood flow and therefore impaired blood flow and organ perfusion are important etiologies in the development of NEC. In addition, the age of storage of blood units, the more likely the occurrence of NEC, and oxidative stress have been mentioned as potential factors. PRC transfusion induces a pro-inflammatory response that may support the pathogenesis of transfusion-associated NEC. 6

PRC transfusion volume is associated with retinopathy of prematurity (ROP) and most studies suggest that iron overload and oxidative stress may be major players in ROP. In addition, the increased delivery of O2 to the retinal vasculature after transfusion with adult RBCs may impair the function of growth factors that regulate retinal vasculature. Again low birth weight and respiratory distress were also independent risk factors for the development of ROP

Multivariate regression analysis found that gestational age and frequency of blood transfusions were independently associated with the risk of developing ROP (Cooke et al, 1993). They reported a 9% increase in the risk of ROP with each transfusion administered (95% CI 1.0–1.18). Hesse et al demonstrated by the same statistical method, that blood transfusion was an independent risk factor for ROP. In this study, the relative risk of developing ROP was 6.4 (95% CI 1.2-33.4) for infants who received 16-45 ml/kg, and 12.3 (1.6-92.5) for those who had received more of 45 ml/kg blood (reference, 0-15 ml/kg). (Mikaniki, 2012)

Figure 6. The process of ROP

Figure 6. The process of ROP

PRC transfusion given before the occurrence of intravascular hemorrhage (IVH) is an independent risk factor for the development of severe IVH. The association between IVH and blood receptivity may be related to volutrauma and damage to weak blood vessels in the germinal matrix. This may be further exacerbated by loss of NO from erythrocytes during storage which would impair capillary vasodilation. 6

In premature infants, red cell transfusion was found to be associated with bronchopulmonary dysplasia (BPD), and an increased number of transfusions was found to be associated with disease severity. Transfusion-associated iron overload and oxidative stress are potential mechanisms linking transfusion to the development of BPD. The association between blood transfusion and BPD may be related to the finding that infants with BPD are usually smaller, require more ventilator support and require more blood sampling, leading to iatrogenic anemia. As a result more blood transfusions will be required to replace the one taken by sampling. This suggests a potential consequence of very low birth weight rather than a direct cause of BPD. However, receiving a blood transfusion and the associated complications caused by it can exacerbate conditions developing from other causes. The main factor in the development of BPD is endotracheal infection. Recent studies in critically ill adults have shown that transfusions with blood stored for more than 14 days are associated with an increase in bacterial infections. Since iron availability is critical for bacterial colonization, and iron-mediated transfusion promoting bacterial infection may be involved in the development of BPD.
In a study conducted by Zhang et al in 2014, the incidence of BPD was significantly higher in the group of infants with transfusion than in the group without transfusion (37.2% vs. 2.1%, P < 0.00001). After adjusting for potential risk factors, the adjusted odds ratio for BPD was 9.80 (95% confidence interval, 1.70–56.36; P = 0.01). This study demonstrated an association between PRBC transfusion and BPD in premature infants.(zhang, 2014)

Neonatal platelet transfusion
Thrombocytopenia, generally defined as a platelet count less than 150 × 109/L, is the second most common (after anemia) haematological disorder in infants admitted to neonatal intensive care units (NICUs). It affects 18-35% of all patients admitted to the NICU and approximately 70% of very low birth weight (ELBW) infants with a birth weight of less than 1,000 grams.

The etiology of thrombocytopenia is very diverse. Clinically, a distinction is often made between “early onset” (≤3 days of life) and “late-onset” (≥4 days of life) neonatal thrombocytopenia. Impaired intra-uterine growth, pregnancy-induced hypertension or diabetes, perinatal infection, and transplacental passage of maternal allo- or autoantibodies are frequently associated with early-onset thrombocytopenia. Late onset neonatal thrombocytopenia is most often caused by bacterial infection or necrotizing enterocolitis

Hyporesponsiveness of neonatal platelet activation and aggregation to most agonists (including ADP, epinephrine, collagen, thrombin, and thromboxane analogues), compared with adult platelets. In particular, the lack of response to epinephrine is explained by the presence of fewer 2-adrenergic receptors on neonatal platelets, a reduced response to collagen likely a consequence of impaired calcium mobilization, and reduced neonatal thromboxane. Decreased expression of PAR-1 and PAR-4 receptors on neonatal platelets explains the decreased response to thrombin. While this platelet hyporeactivity is expected to produce a bleeding tendency. The presence of compensatory factors in the neonatal blood that promote clot formation and perfect balance of neonatal platelet hyporeactivity, leads to normal BT and CT. These compensatory factors include a high hematocrit in neonatal blood, neonatal red cells with a high MCV, and the predominance of the ultralong polymer von Willebrand Factor, which is also present at high concentrations in neonatal plasma. Thus, neonatal platelet hyporeactivity should not be considered as a deficiency of clotting factors or factors that lead to bleeding tendencies, but rather a part of a balanced and unique hemostatic system. 9

Posted in ANEMIA | Tagged , , , , , , , , , , | Leave a comment

USE OF CYCLOSPHAMIDE IN AUTOIMMUNE HEMOLYTIC ANEMIA

USE OF CYCLOSPHAMIDE IN AUTOIMMUNE HEMOLYTIC ANEMIA

PRELIMINARY
Autoimmune hemolytic anemia is anemia that arises due to the formation of autoantibodies against erythrocytes, causing destruction/hemolysis of erythrocytes.
The classification of autoimmune hemolytic anemia based on the nature of the antibody reaction is divided into 2 groups, namely warm type hemolytic anemia (warm AIHA) and cold type hemolytic anemia (cold AIHA).
The incidence of autoimmune hemolytic anemia in children is relatively rare, which is about 1/80,000 children in the general population. The frequency of warm-type autoimmune hemolytic anemia is more frequent, which is about 50-70% of all autoimmune hemolytic anemias

The causes of autoimmune hemolytic anemia are mostly idiopathic. Other causes are secondary to lymphoploriferative disease (non-Hodgkin’s lymphoma, chronic lymphocytic leukemia), autoimmune diseases (systemic lupus erythematosus), and infectious diseases.

Clinical symptoms of autoimmune hemolytic anemia include pallor, jaundice, and hepatosplenomegaly. If there is severe anemia, it will be found shortness of breath, tachycardia, and impaired consciousness
The diagnosis of autoimmune hemolytic anemia is by finding evidence of hemolysis (destruction of erythrocytes, increased hemoglobin catabolism, increased erythropoiesis activity) and evidence of autoantibodies against erythrocytes (positive Coomb’s test). 1,4,5

Because it is a rare case, the management of autoimmune hemolytic anemia is challenging, mainly due to the absence of evidence-based consensus guidelines and limited clinical trial studies to define standard therapy. Currently, first-line therapy is corticosteroids. Alternative second-line therapy is the administration of strong immunosuppressives (cyclophosphamide, cyclosporine A, azathioprine, mycophenolate mofetil), immunoglobulins. The third line of therapy is splenectomy.6,7

This paper will report a case of autoimmune hemolytic anemia in a child who was treated with high-dose methylprednisolone, but did not show improvement. Treatment was then replaced with high-dose cyclophosphamide and showed improvement with increasing hemoglobin levels.

CASE REPORT
AP, daughter, age 10 years 5 months admitted to RSUP Dr. Wahidin Sudirohusodo on September 18, 2013 with a working diagnosis of hemolytic anemia and malnutrition.
History
Paleness was noticed since 5 days before admission to the hospital. No fever. No cough. No vomiting. No stomach pain. There is weakness, fatigue, and heart palpitations. Children are lazy to eat and drink. CHAPTER = normal, yellow color, BAK = smooth impression, color like strong tea. History of frequent fever, no history of spontaneous bleeding, no history of previous drug consumption, no history of visiting malaria endemic areas, history of transfusion there was no previous blood, no history of malignancy in the family, history of being treated at Haji General Hospital for 1 day and planned to do a blood transfusion, but it was canceled due to incompatibility results then the patient was referred to Dr. Wahidin Sudirohusodo.

Physical examination

The child appears to be seriously ill, malnourished (weight 24 kg, PB 134 cm), aware of GCS 15 (E4M6V5). Blood pressure 100/60 mmHg; pulse 124 x/minute, regular, contains; respiration 32 x/minute, temperature 37.10 Celsius, puberty status A1M1P1. There is pallor, there is jaundice. No enlarged lymph nodes. Examination of chest wall inspection within normal limits. On auscultation of the chest, there were no crackles or wheezing, pure regular I/II heart sounds, no heart murmurs. Abdominal examination of the liver and spleen was not palpable. There were no bleeding manifestations either spontaneous or provocation.

Laboratory examination
Routine blood (18/9/2013):
Hb 3.4 g/dL, hematocrit 7.1%, leukocytes 7100/mm3, platelets 183,000/mm3, erythrocytes 0.6 million/mm3. MCV 118.3 fL, MCH 56.7 pg, MCHC 47.9 gr/dL. Count types: neutrophils 89.5%, eosinophils 0.1%, basophils 0.1%, monocytes 1.1%, lymphocytes 9.2%.
Reticulocytes: 19.7%
Peripheral blood smear (18/9/2013):
Erythrocytes: normocytic normochromic, anisopoikilocytosis, ovalocytes (+), cell fragments (+), stomatocytes (+), target cells (+), polychromasia (+), inclusion objects (-), normoblasts (+)
Leukocytes: sufficient number, PMN>lymphocytes, toxic granulation (+), hypersegmentation (+), young cells (-)
Platelets: sufficient number, normal morphology
Impression: normochromic normocytic anemia with hemolytic signs accompanied by leukocytes, signs of infection
Suggestion: bilirubin I/II, Coomb’s test
Routine urine (18/9/2013)
Yellow color, pH 7, Bj 1.005, protein (-), glucose (-), bilirubin (-), urobilinogen (++), blood (-), erythrocyte sediment 1-2, leukocyte sediment 0-1.
Blood chemistry:
Total Bilirubin : 2.7 mg/dL; direct bilirubin 0.4 mg/d; indirect bilirubin 2.3 mg/dL; albumin 4.1 g/dL; SGOT 31 U/L; SGPT 15 U/L
DDR : negative
Coomb’s test: positive

DEFINITIVE DIAGNOSIS
– Autoimmune hemolytic anemia
– Malnutrition

MANAGEMENT
Medical Care
– Oxygen via nasal cannula 2 liters/minute
– IVFD dextrose 5% 20 drops/minute
– Methylprednisolone 20 mg/kg/day

Nutritional care
– Regular food:energy 1440 kcal, protein 54 grams.
Follow-up observations to:
3: KU: weak, vital signs T= 90/60 mmHg, N= 124x/minute, P= 38x/minute, S = 37.10 Celsius. No fever, cough, or shortness of breath. There is still weakness, fatigue, and heart palpitations. There is still pallor, there is still jaundice, no organ enlargement.
Medical care: Oxygen nasal cannula 2 liters/minute, IVFD dextrose 5% 20 drops/minute, dose of methylprednisolone is increased to 30 mg/kgBW/day = 360 mg/intravenous
Nutritional care: Regular food 1440 kcal energy, 54 grams protein.

Laboratory :
routine blood : WBC 7200; Hb 2.5 g/dL; RBC 0.46 million/uL; HCT 5.6%, PLT 172,000; MCV 121.7 fl; MCH 47.8 pg; MCHC 39.3 g/dL, neut 55.9%, Lymphocytes 35.3%, mono 6.7%, eos 0.3%, basophils 1.8%
Coomb’s test: positive 2, the results of cross matching showed major and minor incompatibility (+2) and IgG was found in DAT.
6: KU: weak, vital signs T= 100/70 mmHg, N= 116x/min, P= 28x/min, S = 36.80 Celsius. There is still pallor and jaundice, no organ enlargement.
Medical care: Supportive therapy is continued, methylprednisolone is replaced with cyclophosphamide 100-200 mg/m2/day = 175 mg/intravenous

Nutritional care: Regular food 1440 kcal energy, 54 grams protein.
Routine blood: WBC 11,090; Hb 2.5 g/dL; RBC 0.48 million/uL; HCT 6.4%, PLT 170,000; MCV 133.3 fl; MCH 52.1 pg; MCHC 39.1 g/dL, neut 63%, lymphocytes 28.1%, monocytes 8.0%, eos 0.5%, basophils 0.4%.
Coomb’s test: positive 2, there are still major and minor incompatibility (+2).
9: KU: weak, vital signs T= 100/70 mmHg, N= 116x/min, P= 28x/min, S = 36.80 Celsius. There is still pallor and jaundice, no organ enlargement.

Medical care: Continued supportive therapy, continued cyclophosphamide 100-200 mg/m2hr = 175 mg/intravenous
Nutritional care: Regular food 1440 kcal energy, 54 grams protein.
Routine blood: WBC 7500; Hb 3.6 g/dL; RBC 0.48 million/uL; HCT 6.4%, PLT 167,000; MCV 147.8 fl; MCH 39.1 pg; MCHC 26.5 g/dL, neut 67%, lymphocytes 22.7%, monocytes 7.0%, eos 2.0%, basophils 0.4%
Coomb’s test: positive 2, there are still major and minor incompatibility (+2).
12 : KU : weak, vital signs T= 100/70 mmHg, N= 106x/min, P= 24x/min, S = 36.80 Celsius. There is still pallor, no jaundice, no organ enlargement.

Medical care : Cyclophosphamide was discontinued after 6 days of administration, then continued with oral methylprednisolone 1 mg/kgBW/day = 24 mg = 2-2-2/oral (4 mg tablets)
Nutritional care: Regular food 1440 kcal energy, 54 grams protein.
Routine blood: WBC 4900; Hb 4.0 gr/dL; RBC 0.48 million/uL; HCT 11.8%, PLT 150.000; MCV 120 fl; MCH 40.5 pg; MCHC 33.8 g/dL, neut 66.8%, lymphocytes 21.1%, monocytes 8.7%, eos 2.9%, basophils 0.5%
Reticulocytes: 3.1%
15 : KU : weak, vital signs T= 100/70 mmHg, N= 96x/minute, P= 20x/minute, S = 36.50 Celsius. No pallor and jaundice.
Medical care : Methylprednisolone tablets 2-1-1/oral (tapering off)
Nutritional care: Regular food 1440 kcal energy, 54 grams protein.
Routine blood: WBC 8200; Hb 9.5 gr/dL; RBC 4.03 million/uL; HCT 29.2%, PLT 297,000; MCV 72 fl; MCH 23.5 pg; MCHC 32.4 g/dL, neut 39.7%, lymphocytes 50.3%, monocytes 7.7%, eos 1.2%, basophils 1.1%
The patient was allowed to go home, and was recommended for control at the pediatric polyclinic.

DISCUSSION
Autoimmune hemolytic anemia in children is associated with several diseases such as: immunodeficiency syndromes, malignancies, and systemic autoimmune diseases.1 Sometimes no underlying disease can be detected as in this case.

Autoimmunity is a condition in which a person’s immune system responds to itself (self response) resulting in disease. To understand the pathogenesis of the autoimmune response, it is necessary to understand the mechanism of the immune system’s self-tolerance. Failure of tolerance due to induction of foreign antigens will result in autoimmunity. A B or T cell that does not respond to a “self” antigen is called tolerance (normal), but B or T Cell Tolerance is also referred to if it does not respond to a foreign antigen (abnormal). For autoimmune speech, tolerance is self-tolerance. B cells or T cells whose receptors are in accordance with self antigens should not develop so that there is no immune response to self antigens, but for some reason these T cells or B cells actively respond to self antigens, autoimmune diseases occur.

Antibody-mediated destruction of erythrocytes occurs through activation of the complement system, activation of cellular mechanisms, or a combination of both. Overall activation of the complement system will cause the destruction of the erythrocyte cell membrane and intravascular hemolysis occurs. IgM is called cold-type agglutinin, because these antibodies bind to polysaccharide antigens on the surface of red blood cells at temperatures below body temperature. 2,3,4

 

Figure 1. Pathomechanisms of intravascular and extravascular hemolysis

Figure 1. Pathomechanisms of intravascular and extravascular hemolysis

IgG antibodies are called warm-type agglutinins because they react with erythrocyte cell surface antigens at normal body temperature. If lysed blood cells are synthesized with IgG that is not bound to complement components but no further complement activation occurs, then the red blood cells will be destroyed by reticuloendothelial cells. This erythrocyte destruction process is mainly mediated by IgG-FcR which will cause phagocytosis. This causes extravascular hemolysis.

The diagnosis of autoimmune hemolytic anemia in this case was based on history, physical examination, and investigations. In the anamnesis it was found that there was pallor which was noticed since 5 days before admission to the hospital. On physical examination, he was pale and icterus. Investigations revealed anemia with hemoglobin levels of 3.6 mg/dL, reticulocytosis of 19.7%, an increase in indirect bilirubin of 2.3 g/dL, the presence of normoblasts on the peripheral blood smear, the presence of urobilinogenuria, and a positive Coomb’s test. This case is classified as warm type autoimmune hemolytic anemia because hemolysis occurs at body temperature > 37o Celsius, evidence of extravascular hemolysis, and the presence of IgG antibodies on cross-matching examination. In addition, an alloimmune process was found in this case, because the results of major and minor incompatibility were obtained on cross matching examination.

Immunosuppressive therapy with corticosteroids is the first-line therapy for warm-type AIHA. A response is seen in about 80% of cases.7 Studies conducted by Naithani et al in 2007 and Nazan et al in 2010 showed that corticosteroid therapy is effective in the treatment of autoimmune hemolytic anemia.7,11 (Level III, recommendation A). Research by Gurgey in 1999 that corticosteroid therapy is effective in acute cases, while in chronic cases the response varies.12 (Level III, recommendation C). Research by Yetgin et al in 2007 that high-dose corticosteroids are more effective than conventional doses in haematological disorders.13 (Level I, recommendation A). Research by Gehrs in 2002 that the response to steroid therapy will usually be seen within 1 week of treatment

Corticosteroids are believed to inhibit Fc receptors from binding to IgG so that phagocytosis by macrophages in extravascular hemolysis can be inhibited,15 while cyclophosphamide is an immunosuppressive agent that suppresses the production of autoantibodies in B lymphocytes and T lymphocytes.
In this case, after administration of high-dose methylprednisolone for 6 days, the patient did not respond to steroids. Seen by a decrease in hemoglobin levels on monitoring to 2.5 g / dl. This means that the hemolysis process is still continuing. So in this case steroid therapy was replaced with cyclophosphamide.

A case report by Panceri et al in 1992 showed that autoimmune hemolytic anemia responded well to cyclophosphamide after no response to steroids.16 (Level III, recommendation C) while Ettore et al reported a good therapeutic response after 10 days of combined steroids and cyclophosphamide. .17 (Level III, recommendation C). A report by Ahmad et al in 2013 that cyclophosphamide is good for refractory autoimmune hemolytic anemia. In his study showed a significant increase in hemoglobin levels after cyclophosphamide therapy. (Level III, recommendation C).6 A study by Moyo et al in 2002 showed that 9 patients with autoimmune hemolytic anemia unresponsive to corticosteroid treatment and other treatments were given cyclophosphamide for 4 days, and it was seen that 6 patients had complete remission without transfusion. blood. (Level III, recommendation C).18
In this case, after administration of cyclophosphamide for 6 days, the patient’s hemoglobin level increased without blood transfusion. The following is a graph showing the increase in hemoglobin levels in our case, after six days of cyclophosphamide administration.

Graph 1. Increased hemoglobin levels after administration of cyclophosphamide

Graph 1. Increased hemoglobin levels after administration of cyclophosphamide

After administration of cyclophosphamide, the reticulocyte level decreased from 19.7% to 3.1%, which means that the hemolysis process began to decrease. The following is a graph showing the decrease in reticulocyte levels in our case after six days of cyclophosphamide administration.

Graph 2. Decreased reticulocyte levels after administration of cyclophosphamide

Graph 2. Decreased reticulocyte levels after administration of cyclophosphamide

 

Several case reports reported an increase in hemoglobin levels after administration of cyclosporine A. In contrast to cyclophosphamide, cyclosporine A only suppresses the production of autoantibodies by T lymphocytes so that in this case the second-line therapy chosen was cyclophosphamide. There are also reports of successful treatment of autoimmune hemolytic anemia using immunoglobulins as second-line therapy. Immunoglobulins function in the blockade of phagocytosis by macrophages because immunoglobulins bind to receptors on macrophages so that they cannot bind to IgG.

In this case, conventional steroid therapy was given to achieve complete remission and prevent relapse. Research by Nathalie et al in 2011 that complete remission mostly occurs in the first month of therapy. Patients with refractory autoimmune hemolytic anemia require approximately 1 to 7 years of therapy. After multivariate analysis, it was found that only IgG/IgG+C3d DAT was significantly associated with complete remission with a Hazard ratio of 0.43; 95% CI 0.21-0.68, p=0.01.12 (Level III, recommendation C). A report by Moyo et al in 2002 that in 6 patients who experienced complete remission after administration of cyclophosphamide none experienced a relapse with a median follow-up of 15 months (range 4 months-29 months).18

Several case reports of autoimmune hemolytic anemia unresponsive to steroids and second-line therapy reported splenectomy. Splenectomy plays a role in blocking the site of destruction of erythrocytes and suppressing the production of B lymphocytes, because the spleen is a lymphoid organ.7 In this case, splenectomy was not considered because with second-line therapy clinical and laboratory improvements have been seen.
The prognosis of this case is dubia, because there has been an increase in hemoglobin levels, although it has not reached normal values. Monitoring and adherence to medication is required for complete remission to occur.

SUMMARY
A case of autoimmune hemolytic anemia has been reported in a girl 10 years 5 months. The diagnosis is based on history, physical examination and investigations. Therapy in this case was high-dose corticosteroids, but no improvement was seen so they were replaced with high-dose cyclophosphamide. Improvement was seen with increasing hemoglobin levels without blood transfusion.

 

REFERENCES

  1. Aljedai A. Immune Haemolytic Anaemia. 2010. Available at http/www.emedicine.com.
  2. Anonim. Autoimmune Hemolytic Anemia. Available at http//www. Wikipedia.com
  3. Ware RE. Autoimmune Hemolytic Anemia in Children. http // www. pathology thread.com
  4. Dhaliwal G. Hemolytic Anemia. San Francisco Veterans Affairs Medical Center/University of California–San Francisco School of Medicine, San Francisco, California Am Fam Physician.2004 Jun 1;69(11):2599-2607.
  5. Shoenfield, Y. Diagnostic Criteria in Autoimmune Disease. Humana Press. 2008.
  6. Ahmad F, Mustofa S. Pulse Cyclophosphamide Therapy in Refractory Warm Autoimmune Hemolytic Anemia: A New Perspective. Indian Journal of Hematology and Blood Transfusion. 2013.
  7. Nazan S, Suar C, Emine Z, Sema A. Management of Autoimmune Hemolytic Anemia in Children and Adolescent : A single Centre Experience. Turk J Hematology 2011; 28 : 198-205.
  8. John Clancy, JR : Basic concepts in Immunology. A student’s survival guide. (Mc.Graw-Hill International editors, 2000).
  9. Karnen G. Baratawidjaja: Immunlogi Dasar, Ed VI, Balai Penerbit Fak Kedokteran UI, 2004.
  10. Abul K.Abbas: Basic Immunology. Second Ed.Saunders Elseier, 2006
  11. Naithani R, Agrawal N, Mahapatra M, et al. Autoimmune hemolytic anemia in children. Pediatr Hematol Oncol 2007;24:309-15.
  12. Gurgey A,Yenicesu I, Kanra T, Azsoylu S, Alyay C, Yetgin S. Autoimmune hemolytic anemia with warm antibodies in children. Retrospective analysis of 51 cases. Turk J Pediatr 1999; 41: 467-471.
  13. Yetgin S, Azsoylu S. Comparison Megadose Methylprednisolone versus Conventional Dose Prednisolone in Hematologic Disorders. J Pediatric Hematol Oncol 2007; 29 (4) : 253-9.
  14. Gehrs BC and Friedberg RC. Autoimmune HemolyticAnemia. Am J Hematol 2002;69:258-71.
  15. Nebe Yaraly, Tunc Fybgyn, Abdurrahman Kara, Feride Duru. Successful management of severe chronic autoimmune hemolytic anemia with low dose cyclosporine and prednisone in an The Turkish Journal of Pediatrics 2003; 45: 335-337.
  16. Panceri R, Fraschini D, Tornotti G, Masera G, Locasciulli A. Succesfull Use of High Dose Cyclophospamide in a Child with Severe Autoimmune Hemolytic Anemia. Hematologica 1992; 77(1) : 76-8.
  17. Ettore B, Giorgio A, Fabio R, Momcilo J, Barbara N. A Persistent Severe Autoimmune Hemolytic Anemia Despite Apparent Direct Antiglobulin Test Negativization. Hematologica Journal 1999; 84 : 1043-45.
  18. Moyo VM, Smith D, Brodsky I,Crilley P, Jonas RJ. High Dose Cyclophospamide for Refractory Autoimmune Hemolytic Anemia. Blood 2002; 100(2): 704-6.
  19. Nathalie A, Guy L, Thierry L, Marie P, Gerard M, Yves B, Alain R, Virginia G, Alain F, Caroline T, et al. New Insight into Childhood Autoimmune Hemolytic Anemia : A French National Observational Study of 265 Chilren. Henatologica 2011; 96(5): 655-663.

By :

dr.Besse Sarmila, SpA, Dr.dr.Nadirah Rasyid Ridha,M.Kes,Sp.A(K), Prof.Dr.dr.H.Dasril Daud,Sp.A(K)

PROGRAM PENDIDIKAN DOKTER SPESIALIS

DEPARTEMEN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN

UNIVERSITAS HASANUDDIN

MAKASSAR

Posted in HEMATOLOGY | Tagged , , , , , , | Leave a comment

Cluster of Differentiation 44 (CD44)

Cluster of Differentiation 44 (CD44)

PRELIMINARY
The adhesion molecule CD44 is a transmembrane glycoprotein that plays a role in lymphocyte activation, recirculation, and balance, extracellular matrix adhesion, angiogenesis, cell proliferation, cell differentiation and cell migration and as a receptor for hyaluronic acid. These various biological properties are very useful for the physiological activity of normal cells, but are also associated with the pathological activity of tumor cells. Increased expression of CD44 is associated with poor prognosis of several malignancies such as lung cancer, ovarian cancer, breast cancer, colorectal cancer, gastrointestinal neuroendocrine tumors, and others. In recent years, scientists have focused more on the relationship between CD44 and blood malignancies.

They assumed that CD44 plays an important role in normal myelopoiesis because anti-CD44 antibodies are fundamentally altered in in vitro myelopoiesis in long-term bone marrow culture. In the context of leukemia, studies have shown that it is possible to stop differentiation in some leukemic cells by binding (ligation) to CD44 with specific antibodies, indicating the possibility of recent advances in targeted therapy for CD44 differentiation in leukemia therapy.

CD44 structure and function
The human CD44 gene is located in the forearm on chromosome 11 which consists of at least 20 exons that rotate 50 kilobases of the DNA chain. This gene consists of 2 groups of exons, consisting of exons 1-5 and 16-20 which are expressed simultaneously in all cell types and are standard forms. 10 exon variables (6-15) can be randomly connected via standard exons at the insertion site between exons 5 and 16. Transcription for this gene is via alternative linkage complexes that are generated in functionally distinct isoforms. This type of isoform is divided into peptide units consisting of the extracellular region of the protein, according to the alternative splicing theory that will result in more than 1000 variations of the CD44 molecule overall.

The smallest CD44 molecule, which lacks an intact structure, is standard CD44 (CD44s) which is mainly expressed by basic lymphohematopoietic cells. CD44 is also known as hematopoietic (CD44H). CD44s are formed from the distal extracellular region (including the “ligand-binding sites”), the proximal membrane region, the transmembrane ring region and the cytoplasmic end (figure 1). Including multiple glycosylated sites and chondroitic acid binding sites, the extracellular regions can bind different extracellular matrices. The N-terminal is the region primarily responsible for hyaluronic acid binding.

The transmembrane region is very characteristic of most single-membrane glycoproteins including sites that can interact with hexadecanoic acid. The cytoplasmic sequence can be phosphorylated as a substrate for protein kinase C. As a GTP binding protein, CD44 can bind to GDP substrate and has GTP enzymatic activity so that it can increase the interaction between CD44 and ankirins.2,3,4

The CD44 variant (CD44v) is predominantly expressed on epithelial cells, characterized by a broadly glycosylated amino acid site and a chondroitic acid binding site. Connected continuously or in a septal direction, the exon combinations have different variables and different CD44 molecules. Currently, there are more than 10 kinds of CD44v in several cell lines detected by polymerase chain reaction. 1.2

Alternative splicing is the basis for distinguishing the structure and function of these proteins and may be related to tumor metastasis. After immunological activation, CD44v on T lymphocytes and other leukocytes is transiently regulated. The CD44 isoform includes the last 3 exon products in the region variable (CD44v8-v10), also known as epithelial CD44 or CD44E which is specifically expressed on epithelial cells. The longest CD44 isoform is expressed by a fusion of 8 exons of the variable region (CD44v3-v10) present in keratinocytes. pMeta-1 (CD44v4-v7) and pMeta-2 (CD44v6,v7) are referred to as metastatic CD44 because their cDNA causes transfection, the possibility of metastasis in nonmetastatic tumor cells in mice.

Figure 2 shows the CD44 exon arrangement consisting of constant-region exons and exon variations. Figure 1) epithelial CD44 (CD44v8-v10), 2) keratinocyte CD44 (CD44v3-v10), 3) pMeta-1 (CD44v4-v7), pMeta-2 (CD44v6, v7).

Gambar 1(A) dan 1(B). Struktur CD44

Gambar 1(A) dan 1(B). Struktur CD44

Figure 2. CD44 . exon setup

Figure 2. CD44 . exon setup

CD44 function
CD44 is a Cell Adhesion Molecules (CAMs) which is very important to maintain the stability of the tissue structure. In dynamic situations, cells alter their cell and cell matrix interactions through the properties of CAMs that function as expression modifiers. CAM expression is normally tightly regulated, by controlling cell proliferation, mobility, differentiation, and survival. As an adhesion molecule (binder), CD44 plays a role in many ways for example having a role in maintaining the balance of lymphocyte production, T lymphocyte activation, assisting the attachment between fibroblasts, lymphocytes and extracellular material (ECM) such as hyaluronic acid, chondroitin sulfatase, fibronectin, laminin and collagen. . It also plays a role in signal transmission, updating the composition of interstitial tissue, helping drug absorption and drug sensitivity. In addition, it also plays a role in forming pseudopods and plays a role in cell migration

Lymphocyte extravasation at the site of inflammation consists of several steps, namely recognition and rolling, adhesion, diapedesis, and migration. Rolling of T lymphocytes on the endothelium can be mediated by CD44. The interaction between CD44 on T lymphocytes and hyaluronan on the endothelial surface is sufficient to initiate the rolling process required for extravasation to tissues. This rolling interaction together with chemokines facilitates adhesion, mediated by integrins and their receptors on endothelial cells that trigger diapedesis and migration to sites of inflammation and infection. 2

Gambar 4. Pengaturan signal cascade oleh interaksi hyaluronan dan CD44

Gambar 4. Pengaturan signal cascade oleh interaksi hyaluronan dan CD44

Hyaluronan synthase produces and secretes hyaluronan. This release of hyaluronan interacts multivalently with CD44 to induce or stabilize signaling domains in the plasma membrane. This signaling domain contains receptor tyrosine kinases (ErbB2 and EGFR), other receptors such as TGFβR1, and non-receptor kinases (Scr family), which are oncogenic pathways such as cell proliferation by MAP kinase and P13 kinase. adapter proteins mediate the interaction of CD44 with surface effectors such as RhoA, Rac1, and Ras. Heparan sulfate activates the receptor tyrosine kinase, the c-Met receptor which induces cytoskeletal changes to promote cell motility and invasion, by means of actin filaments joining the CD44 tail via the ERM family or ankirin. All of these activities were influenced by hyaluronan produced by tumor cells (figure 4).

Most epithelial cells, hematopoietic cells, and non-epithelial cells generally produce CD44s. The standard CD44 isoform is produced by all mature blood cell types, mostly by bone marrow precursor mononuclear cells and all CD34+ HPC. The level of each expression varies depending on the origin of the hematopoietic cells and the degree of differentiation. For example, CD44 was high in monocytic cells, moderate levels in polymorphonuclear (PMN) and CD34+ HPC and low in erythrocytes and platelets. CD44-6v and CD44-9v isoforms have been detected in monocytes, macrophages, lymphocytes, and dendritic cells.2,3

CD44 is produced at very high levels in many tumor types, and is associated with the biologic properties of tumors including tumorogenesis, growth, metastasis, and prognosis. It is a definite indicator of tumor severity and disease activity and is also called a metastasis-associated protein. In fact, the spread of metastases involves interactions between tumor cells and endothelial cells, which suggests that CD44 may be involved in the process of metastatic expansion. There are several opinions regarding the relationship between lymphocyte activation and tumor cell metastases including strong invasion, reversible adhesion to cell migration, accumulation and proliferation in lymph nodes and sometimes release into the circulatory system and peripheral tissues. Seiter et al assumed that this similarity might be in view of the general effects of CD44v6, suggesting that the mechanism of CD44v6 in tumor metastasis is similar to that of lymphocyte activation. Tumor cells can cause hidden lymphocytes because overexpression of CD44v6 is eliminated through recognition and killed by the immune system, so tumors can invade lymph nodes and metastasize more easily.2,3

CD44 expression in hematological malignancies
Recent findings have shown that CD44 is overexpressed by hematopoietic cells and is involved in interactions between the bone marrow stromal layer and cells of hematopoietic origin and this overexpression is associated with poor prognosis of some haematological malignancies.

CD44 and Acute Myeloid Leukemia
Bendall et al. compared the expression of the CD44 variant in normal bone marrow, peripheral blood and CD34+ cells of hematopoietic origin generated in blasts from 30 patients with acute myeloid leukemia (AML). Normal bone marrow, peripheral blood and CD34+ origin cells were negative in all variants measured using flow cytometry where exons v3,v4,v5,v6 and v7 were expressed in AML cases. RT-PCR and Southern revealed a more complex pattern of expression of exon variants in leukemic samples compared to normal hematopoietic cells. These data demonstrate a significant increase in the complexity of CD44v expression in cells in AML patients through the surface expression of several variants of the CD44 protein. They suggest that further research should be carried out directly on how the interaction of the leukemia blast with the bone marrow microenvironment changes and its diagnosis, prognosis and therapeutic opportunities. Florian et al analyzed the expression of target antigens on CD34+/CD38- cells in patients with AML, myelodysplastic syndrome, chronic myeloid leukemia, and systemic mastocytosis. Using multi-color flow cytometry, they reported that CD44 was expressed in all patients and that neoplastic stem cells in some myeloid neoplasms produced similar phenotypes including target antigens CD13, CD33 and CD44.4.

CD44 and Acute Lymphocytic Leukemia
It has been reported that high levels of the CD44 variant suggest a poor prognosis in patients with acute lymphocytic leukemia (ALL). Magyarosy et al analyzed CD44v6 expression in the bone marrow of 16 pediatric patients with ALL using immunocytochemistry. They found that the CD44v6 protein epitope was expressed by leukemic cells in 6 cases of ALL patients, particularly in the moderate or high risk group (except in 1 case). The picture is very similar to the observations made in some adult cancer patients and shows that: possible association with poor prognosis. The potential of CD44v6 expression on leukemic cells as a prognostic indicator in pediatric ALL patients should be further evaluated in larger clinical trials. Using oligonucleotide microarray analysis, Oh et al. reported that CD44 was associated with poor prognosis after analyzing tissue infiltration parameters in 86 patients with ALL. 4

Table 1. Expression of CD44 and CD44v in hematological malignancies4

Table 1. Expression of CD44 and CD44v in hematological malignancies4

The role of CD44 in chemotherapy
The role of CD44 in chemotherapy has recently been widely studied. One of them is the administration of doxorubicin. Doxorubicin binds to HNA (π-hyluronan nanocarrier) which then CD44 as a hyaluronan receptor assists the endocytosis process of the HNA-DOX complex into target cells. With a low pH environment protons and doxorubicin are released and cause cell nucleus apoptosis (figure 6).7

Gambar 6. CD44 sebagai reseptor hialuronan membantu proses endositosis kompleks πHNA-DOX masuk ke sel target

Gambar 6. CD44 sebagai reseptor hialuronan membantu proses endositosis kompleks πHNA-DOX masuk ke sel target

CONCLUSION
CD44 is a transmembrane glycoprotein that plays a role in lymphocyte activation, recirculation, and balance, extracellular matrix adhesion, angiogenesis, cell proliferation, cell differentiation and cell migration as well as a receptor for hyaluronic acid.
– This gene consists of 2 groups of exons, consisting of exons 1-5 and 16-20 which are expressed simultaneously in all cell types and are standard forms. 10 variable exons (6-15) can be connected which are variations of the shape of CD44. Alternative splicing is the basis for distinguishing the structure and function of these proteins and is associated with tumor metastasis.
– The level of each expression of CD44 varies depending on the origin of the hematopoietic cells and the degree of differentiation, but the expression of CD44 is lower in normal tissue than in tumor tissue. CD44 as a hyaluronan receptor plays an important role in chemotherapy.

REFERENCES

  1. Naor D, Sionov RV,Ish SD. CD44: Structure, Function, and Association with the Malignant Process. Hebrew University Hadassah Medical School. 1997.241-319.
  2. Liu J, Jiang G. CD44 and Hematologic Malignancies. The Chinese Society of Immunology. 2006. 359-63
  3. Hertweck MK, Erdfelder F, Kreuzer KA. CD44 in hematological Neoplasia. University of Cologne. 2011.493-508.
  4. Sneath RJ, Mangham DC. The Normal Structure and function of CD44 and its role in neoplasia. Journal Mol Pathol. 1998.191-200.
  5. Toole BP. Hyaluronan-CD44 Interactions in Cancer: Paradoxes and Possibilities. Clin Cancer Res in aacjournals. 2009. 7462-8.
  6. Knudson W, Knudson CB,The Hyaluronan Receptor, CD44. Exp Cell Res. 1999.
  7. Jang E et al., π-hyluronan nanocarier for CD44 Targetted and pH-boosted aromatic Drug Delivery. Jurnal Mater Chem Biol. 2013.5686-5693.
Posted in HEMATOLOGY | Tagged , , , , , , , , | Leave a comment

DEFERASIROX WORK MECHANISM

DEFERASIROX WORK MECHANISM

PRELIMINARY
Iron overload causes the most mortality and morbidity. Iron deposition occurs in important organs, especially the heart, liver, and endocrine glands causing tissue damage and ultimately organ dysfunction and failure. Humans do not have a mechanism to express iron overload, so chelation therapy is needed. Iron chelating is a chelating agent that can bind excess iron in the body so that it can be removed from the body. Therapy should be started as soon as possible when iron deposits are sufficient to cause tissue damage, namely after 10-20 transfusions or when ferritin levels increase above 1000 micrograms per liter.

Iron is an important metal for hemoglobin synthesis, oxidation-reduction reactions and cell proliferation, while excess iron will cause organ dysfunction through the production of reactive oxygen species. The amount of iron in the body ranges from 3-4 g, two thirds is in the red blood cells and is recycled by the destruction of erythrocytes; the remainder is stored in the form of ferritin/hemosiderin, while only 1-2 mg of iron is absorbed through the gastrointestinal tract and circulates in the blood. Body iron metabolism is a semi-closed system, and is critically regulated by several factors including hepcidin. In the bloodstream, iron is usually bound to transferrin and most of the iron bound to transferrin is used by the bone marrow for erythropoiesis. Due to the absence of an active mechanism in the body to excrete iron, a progressive accumulation of body iron is likely to occur as a result of prolonged transfusion in patients with thalassemia. 3

It has long been known that deferoxamine (desferal) is the standard reference for iron chelation therapy in thalassemia patients, but in several studies, the results of low levels of adherence to the use of these drugs have been found. Currently, the latest chelation therapy with oral preparations has been developed, namely deferasirox (trade name: Exjade). It is hoped that with the oral chelation therapy, the patient’s level of compliance in taking the drug will be better and can reduce the effects of morbidity and mortality due to iron overload in thalassemia patients. This paper will discuss deferasirox (Exjade) as iron chelation therapy. 3

Deferasirox
Deferasirox belongs to the tridentate iron group. 2 molecules of deferasirox bind 1 molecule of iron. Deferasirox contains inactive ingredients such as lactose monohydrate, crospovidone, povidone (K30), sodium lauryl sulfate, microcrystalline cellulose, silicon dioxide and magnesium stearate. deferasirox is an orally active iron binder consisting of more than 700 components. The ICL 670 component is the result of a combination of modern chemical development programs with traditional chemical development techniques. 3

Derairox mechanism of action
Deferasirox has a very high affinity and specificity for Fe3+. The active molecule of deferasirox is highly lipophilic and has a relatively smaller molecular weight compared to deferoxamine due to its ability to enter cells and remove iron. Together with its long enough presence in plasma, it has the potential to maximize the chelating effect of stored iron at relatively small, easy-to-reach levels, maximize clearance of stored iron and minimize uncontrolled iron storage.

Excess iron is found in 2 forms, namely LPI (labile plasma iron) and LCI (labile cell iron). LPI is present in the circulation, namely Fe that is not bound by transferrin, while LCI is intracellular Fe that is not bound to ferritin or hemosiderin. Deferasirox works to bind LPI and LCI because of its hydrophilic nature so that it can penetrate intracellularly. As much as 90% of deferasirox is excreted in the feces.

Excessive erythropoiesis, anemia of chronic disease, and hypoxia will increase intestinal iron absorption. When serum transferrin increases to 70%, ferritin will be damaged and cause hemosiderin to increase and free iron species will be found in plasma which will increase reactive oxygen species which will cause tissue damage, organ dysfunction, and death. Deferasirox acts on the cytosolic ferritin iron. Binds iron and excretes it in the urine

Figure 1. Binding of free iron species with iron chelate.

Figure 1. Binding of free iron species with iron chelate.

Figure 2. The mechanism of action of iron chelation in preventing ROI and ROS

Figure 2. The mechanism of action of iron chelation in preventing ROI and ROS

Gambar 3. Mekanisme kerja kelasi besi melalui kompartemen subselular yang berbeda

Gambar 3. Mekanisme kerja kelasi besi melalui kompartemen subselular yang berbeda

One dose of oral deferasirox is more potent than one subcutaneous non-continuous dose of desferoxamine and 10 times more potent than one oral dose of deferipron.

The potential and specific ability of deferasirox to mobilize tissue iron and to increase its excretion has been demonstrated in several animal studies. Administration of single and multiple doses of iron in experimental rats and guinea pigs increased iron excretion, especially in feces. Administration of deferasirox for a long time, namely in rats for 12 weeks and guinea pigs for 39 weeks, showed a significant decrease in iron levels in the liver. This indicates that the main storage site for iron in the body is the target organ of deferasirox. 2.6

SUMMARY
Deferasirox has a relatively smaller molecule and is lipophilic so that in addition to binding to labile plasma iron (LPI), namely Fe which is not bound to transferrin, it is also able to penetrate intracellularly which is able to bind labile cell iron (LCI), namely Fe which is not bound to ferritin or hemosiderin. . LPI and LCI will form ROI and ROS which eventually causes cell death, so that by binding LPI and LCI by deferasirox, ROI and ROS can be prevented. However, if ROI and ROS have been formed, deferasirox no longer works. 90% of deferasirox is excreted in the feces.

REFERENCES

  1. Kohgo,Y.et al. Body iron metabolism and pathophysiology of iron overload. 2008, International Journal of Hematology, Vol. 88, pp. 7-15
  2. Spesific iron chelator determine the route of ferritin degradation. Blood. 2009:4546-51.
  3. Taher A, Capellini. 2007.Update on the Use of Deferasirox in the Management of Iron Overloaded. Therapeutics and Clinical Risk Management.
  4. Cabanthick ZI. Nontransferin bound iron and labile plasma iron in relation to iron toxicity. Institute of Life Science.San Fransisco. 2014.
  5. Rahmilewitz EA, Giardina PJ. How I treat thalassemia. Blood. 2010:3479-86
  6. Debaun, MR. Vichinsky Elliot, Hemoglobinopathy, Thalassemia. Nelson Textbook of Pediatrics, 18 th edition, 2007.

By :

dr.Mahirina Marjani, SpA, Dr.dr.Nadirah Rasyid Ridha,M.Kes,Sp.A(K), Prof.Dr.dr.H.Dasril Daud,Sp.A(K)

PROGRAM PENDIDIKAN DOKTER SPESIALIS

DEPARTEMEN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN

UNIVERSITAS HASANUDDIN

MAKASSAR

Posted in Uncategorized | Tagged , | Leave a comment

COMPONENTS OF IRON IN THE BODY

COMPONENTS OF IRON IN THE BODY

Iron is a vital element that is needed by the body for oxygen transport, oxidative metabolism, and cell growth and proliferation.

Iron consumed from food will be absorbed in the duodenum and to a lesser extent in the jejunum. About 5 to 15% will be absorbed in the form of Fe2+. In the intestine Fe2+ is oxidized to Fe3+, then Fe3+ binds to apoferritin which is then transformed into ferritin and stored in the form of ferritin. Some more Fe2+ is released into the blood plasma. In the plasma Fe2+ is oxidized to Fe3+ and binds to transferrin. Transferrin transports Fe2+ into the bone marrow to combine to form hemoglobin. Transferrin transports Fe2+ to iron stores in the liver, bone marrow, spleen, and RES organs. Then it is oxidized to Fe3+. This Fe3+ combines with apoferritin to form ferritin which is then stored, the iron contained in plasma is balanced with the stored form.

Figure 1. Iron metabolism in the body

Figure 1. Iron metabolism in the body

 

Components of iron in the body
The iron component in the body consists of 3 forms of compounds, namely functional compounds in metabolism and enzymatic in the form of hemoglobin, myoglobin, enzymes, iron reserves in the form of ferritin and hemosiderin, and transport compounds in transferrin.

Iron in hemoglobin
Most of the iron is bound in hemoglobin, which is about 65-80%, which functions to transport oxygen for metabolic purposes in tissues. Hemoglobin is a metalloprotein consisting of globin, apoprotein, and 4 heme groups. The globin chain consists of 4 linked to each other. Tetramer consists of two pairs of different polypeptide subunits namely , , , . Each sub unit has a molecular weight of approximately 16,000 Daltons. At the center of the molecule there is a heterocyclic ring known as a porphyrin that binds an iron atom which is the oxygen binding site. Overall hemoglobin has a capacity of four oxygen molecules. Hemoglobin liberates O2 to the tissues and transports CO2 and protons to the lungs.1,3

Figure 2. Iron bound to hemoglobin

Figure 2. Iron bound to hemoglobin

Figure 3. The role of iron in the formation of hemoglobin

Figure 3. The role of iron in the formation of hemoglobin

Iron in myoglobin
Myoglobin is an oxygen-carrying protein found in muscle, where myoglobin acts as an oxygen reservoir and facilitates the diffusion of oxygen through cells. Myoglobin has 2 molecular components, a single polypeptide chain containing 153 amino acid residues with a molecular weight of 17,600, and a heme group, which contains iron. The heme of myoglobin is known as a prosthetic group because it is a non-protein organic molecule that is closely related to a polypeptide. The binding of the iron atom to a heme involves the four nitrogens of the pyrrole ring. The bound iron can form two additional bonds, one on each side of the heme plane, called the fifth and sixth coordination positions. At the sixth coordination position, ferrous iron in myoglobin binds one oxygen molecule. 3.4

There is 3.5% iron bound to myoglobin. Found in high concentrations in bone and heart muscle. The folding of the globin chain forms a gap that is almost filled with the heme group. Fe2+ ​​has a high affinity for oxygen and is oxidized unidirectionally to form Fe3+. Fe3+ cannot bind oxygen. the non-covalent interaction between the amino acid site and the non-polar porphyrin ring containing the oxygen-binding site increases the affinity of Fe2+ for O2. The increased affinity protects Fe2+ from oxidation and allows reversible O2 binding. All of the amino acids that interact with nonpolar heme except for two histidines, bind directly to the iron atom of the heme and the other histidine stabilizes the oxygen binding site. In high O2 conditions, myoglobin binds a lot of O2 but in low O2 conditions, myoglobin releases O2 which is used in muscle mitochondria to produce ATP aerobically.

Figure 4. Structure of myoglobin

Figure 4. Structure of myoglobin

 

Iron transported in transferrin
Transferrin is a B1 globulin which is a glycoprotein with a molecular weight of 80,000 – 90,000 daltons, consisting of a single-chain polypeptide with 679 amino acids in two homologous domains. The N-terminal and C-terminal each have one binding site for Fe3+. One transferrin molecule binds 2 Fe3+ atoms. Transferrin will bind to the transferrin receptor. Each transferrin receptor binds to 2 transferrin molecules
Transferrin is mainly synthesized by liver parenchyma cells, to a lesser extent in the brain, ovaries, and T lymphocytes. Transferrin has a half-life of 8-11 days.

Figure 4. Fe bound to transferrin5

Figure 4. Fe bound to transferrin5

Iron in cytochrome enzymes
Cytochromes are electron-transferring proteins that contain heme as a prosthetic group. Cytochrome is a type of carrier protein that contains a heme group whose iron atom is isolated between Fe3+ and Fe2+. The structure consists of 3 helix, and the heme structure as a place for Fe. Various cytochromes are found in the respiratory chain, including cytochrome oxidase (aa3), b5, c, and P450. Cytochromes play a role in electron transfer. The electron transport chain is the final stage of the aerobic respiration reaction. Electron transport takes place in the cristae (inner membrane) of the mitochondrion. Molecules that play an important role in this reaction are NADH and FADH2 which are produced in the process of glycolysis, oxidative decarboxylation, and the Krebs cycle. 1.6

Figure 5. Cytochrome structure.

Figure 5. Cytochrome structure.

Iron in reserve form as ferritin and hemosiderin
Ferritin is one of the proteins that are important in the process of iron metabolism in the body. About 20 – 35% of the total amount of iron in the body is in the form of iron stores (depot iron), in the form of ferritin and hemosiderin. Under normal conditions, iron stores consist of 65% ferritin and 35% hemosiderin. 5

Ferritin is a protein complex that is globular in shape, has 24 protein subunits that compose it with a molecular weight of 450 kDa, found in all cells, both in prokayotic cells and in eukaryotic cells. Under normal circumstances, only a small amount of ferritin is present in human plasma. The amount of ferritin in plasma describes the amount of iron stored in our body. When viewed from the crystal structure, one ferritin monomer has five helix constituents, namely blue helix, orange helix, green helix, yellow helix and red helix where the Fe ion is in the middle of the five helix. In humans, the subunits that make up ferritin are of two types, namely Type L (Light) Polypeptide and Type H (Heavy) Polypeptide, which have molecular weights of 19 kD and 21 kD, respectively. Type L symbolized by FTL is located on chromosome 19 while Type H symbolized by FTH1 is located on chromosome 11. Each ferritin complex can store approximately 3000 – 4500 Fe3+ ions in it.

Ferritin and hemosiderin are mostly found in the spleen, liver and bone marrow. Ferritin is a water-soluble intracellular protein, which is an acute phase protein. Hemosiderin is the body’s iron reserves derived from partially degraded ferritin, found mainly in the bone marrow, and is insoluble in water. Under normal conditions, ferritin stores iron in the intracellular which can later be released back for use as needed. Serum ferritin is a reliable and sensitive parameter for determining iron stores in healthy people

Free iron is toxic to cells, because free iron is a catalyst for the formation of free radicals from Reactive Oxygen Species (ROS) through the Fenton reaction. For this reason, cells form a self-protection mechanism, namely by making iron bonds with ferritin

Figure 7. Ferritin and hemosiderin in Fe5 . metabolism

Figure 7. Ferritin and hemosiderin in Fe5 . metabolism

Iron balance in the body
The balance of iron in the body must be maintained so that anemia does not occur. Every day the turnover of iron is 35 mg, but not all of it must be obtained from food. Most of which is as much as 80-90% obtained from the breakdown of old erythrocytes, which are recycled to be used again by the bone marrow to form red blood cells. Factors that affect the balance of iron in the body are the food consumed, the number of erythrocytes in the body, the amount of oxygen in the body, and the influence of drugs. Ferritin is one of the keys that regulate iron hemostasis. Fe3+ stored in ferritin will be released again if the body needs it.1

Conclusion
Iron is needed by the body for oxygen transport, oxidative metabolism, and cell growth and proliferation. Iron in the body consists of functional compounds in metabolic and enzymatic, transport and storage forms of iron. Functional compounds that are bound to hemoglobin, myoglobin, and cytochromes. While the reserve components consist of ferritin and hemosiderin which are degradation of ferritin. Most components are bound to hemoglobin which functions to transport O2, while myoglobin is a protein that stores O2 reserves. While the reserve component is more in the form of ferritin than hemosiderin. Transport of iron is carried out by transferrin which is the least amount.

References
1. Nadadur SS. Iron transport and homeostasis mechanisms. Medical India Journal. 2008. 533-39
2. Knutson M. Iron metabolism in the reticuloendothelial system. Molecular biology biochemistry. 2003.61-88.
3. Patil NN. Hemoglobin: Structure, function, and degradation. 2011
4. Arkhipov A. Case study: myoglobin theoretical biophysics group. 2008. accessed from http://www.ks.uiuc.edu.
5. Thorstensen K, Romslo I. The role of transferrin in the mechanism of cellular iron uptake. Biochemical Journal. 1990. 1-10.
6. The electron transport chain. available from https://www.tamu.edu.2003.
7. Storage iron metabolism. Accessed from http://www.omicsonline.org. 2012.

 

By :

dr.Mahirina Marjani, SpA, Dr.dr.Nadirah Rasyid Ridha,M.Kes,Sp.A(K), Prof.Dr.dr.H.Dasril Daud,Sp.A(K)

PROGRAM PENDIDIKAN DOKTER SPESIALIS

DEPARTEMEN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN

UNIVERSITAS HASANUDDIN

MAKASSAR

Posted in ANEMIA | Tagged , , , , , , , , , | Leave a comment

HEMOSTASIS PRIMER

HEMOSTASIS  PRIMER

A. INTRODUCTION
Maintenance of blood fluidity in the vascular system is an important physiological process in humans. The term ‘hemostasis’ refers to the normal response to injury to a blood vessel by forming a clot that serves to limit bleeding. The important components of fluidity, hemostasis and thrombosis are the blood flow generated by the cardiac cycle, the vascular endothelium and the blood itself. Under normal physiological conditions there is a delicate balance (eucoagulability) between the pathological states of hypercoagulability and hypocoagulability in the circulation.
The hemostatic system reflects the balance between procoagulant and anticoagulant mechanisms associated with the process for fibrinolysis. The five main components involved are platelets, coagulation factors, coagulation inhibitors, fibrinolysis and blood vessels.

Astrup in 1958 first described the phenomenon of the so-called hemostatic balance. Hemostasis in which clots that form in response to an injury are then regulated for self-destruction by stimulating fibrinolytic activity. This concept has been observed whereby blood has a strong tendency to clot in intact vessels so that the blood requires a primary antithrombotic system to prevent clot formation. Under normal circumstances, endothelial cells present a non-thrombogenic surface that does not attract plasma proteins and blood cells.
Under normal physiological conditions, platelets are not attracted by endothelial cells and no adhesive protein is present in the surrounding plasma. Excessive activation of hemostatic mechanisms is prevented by endothelial protective factors, inhibitors of the coagulation system and effects of blood flow.

B. PRIMARY HEMOSTASIS
Primary hemostasis occurs when there is an injury to a blood vessel. This hemostasis involves the tunica intima vascular and platelets. Hemostasis at this stage is rapid but does not last long. Therefore, if primary hemostasis is not sufficient to compensate for the wound, it will proceed to secondary hemostasis. 7.8

C. Platelets
1. Platelet production
Platelets are produced in the bone marrow through the fragmentation of the cytoplasm of megakaryocytes. The precursors of megakaryocytes-megakaryoblasts arise through the process of differentiation from hemopoietic stem cells. Megakaryocytes undergo maturation by synchronous endomitotic nuclear replication, increasing the cytoplasmic volume as the nuclear lobes increase to multiples of two. At various stages in its development, the cytoplasm becomes granular and platelets are released. The production of platelets follows the formation of microvesicles in the cytoplasm of the cell which fuse to form the platelet boundary membrane. Each megakaryocyte is responsible for producing about 4000 platelets. The time interval from the differentiation of human stem cells to the production of platelets ranges from 10 days.

Gambar 1. Hemotopoesis trombosit5

Gambar 1. Hemotopoesis trombosit5

Thrombopoetin is a major regulator of platelet production and is produced by the liver and kidneys. Platelets have receptors for thrombopoietin (C-MPL) and remove it from the circulation, therefore thrombopoietin levels are highest in thrombocytopenia due to bone marrow aplasia and vice versa. Thrombopoetin increases the number and rate of maturation of megakaryocytes. Platelet counts begin to increase 6 days after initiation of therapy and remain high for 7-10 days. Normal platelet count is about 250 x 109/l (range 150-400 x 109 and normal platelet life span is 7-10 days. These cells play an important role in hemostasis to close wounds. Formation of hemostatic plug occurs through several stages, namely platelet adhesion , aggregation and release reactions.3.5

2. Platelet structure
In the inactivated state, platelets are biconvex discs with a diameter of 2-4µm and a volume of 7-8fl. The external sheath of platelets is thicker and denser than cells and contains many glycoproteins that function as receptors. Glycoprotein I and V are receptors for thrombin, glycoprotein Ib is a receptor for Von Willebrand factor while glycoprotein II b and III a are receptors for fibrinogen.

Gambar 2. Ultrastruktur Trombosit5

Gambar 2. Ultrastruktur Trombosit5

Ultrastructurally, platelets can be divided into peripheral zone, sol gel zone and organella zone. The peripheral zone consists of the glycocalyx, an extra membrane located on the outermost part; Inside is the plasma membrane and deeper there is an open canal system. The sol gel zone consists of microtubules, microfilaments, a dense tubular system (containing adenine and calcium nucleotides). In addition there is also thrombostenin, a protein important for contractile function. The organelle zone consists of dense granules, mitochondria, granules and organelles (lysosomes and endoplasmic reticulum). Dense granules contain and release the nucleotides adenine, serotonin, catecholamines and platelet factor. While the granules contain and release fibrinogen, PDGF (platelet-derived growth factor), lysosomal enzymes. There are 7 platelet factors that have been identified and their characteristics are known. Two of them are considered important, namely PF3 and PF4.

Surface glycoproteins are very important in platelet adhesion and aggregation reactions which are the initial events leading to the formation of a platelet plug during hemostasis. The plasma membrane invaginates into the interior of the platelet to form an open (canalicular) membrane system that provides a large reactive surface from which plasma coagulation proteins are selectively absorbed. Membrane phospholipids are essential in the conversion of coagulation factor X to Xa and prothrombin (factor II) to thrombin (factor IIa).

Inside the platelets there are calcium nucleotides, especially Adenosine diphosphate (ADP) and adenosine triphosphate (ATP), and serotonin which is contained in the granules of electrons. Specific granules (more common) contain heparin antagonists, platelet derived growth factor (PDGF), -thromboglobulin, fibrinogen, vWF and other clotting factors. Dense granules are less numerous and contain ADP, ATP, 5-hydroxytryptamine (5-HT), and calcium.
Other specific organelles include lysosomes containing hydrolytic enzymes and peroxisomes containing catalase. During the release reaction, the granular contents are expelled into the canalicular system.3,4
3. Platelet function

The main function of platelets is the formation of a mechanical plug during the normal hemostatic response to vascular injury. Without platelets, spontaneous blood leakage can occur through small blood vessels. Platelet reactions in the form of adhesion, secretion, aggregation, and fusion as well as its procoagulant activity are very important for its function

D. ENDOTELAL CELL
Endothelial cells play an active role in maintaining vascular integrity. These cells produce a basement membrane that normally separates collagen, elastin and fibronectin in the subendothelial connective tissue from circulating blood. Loss or destruction of the endothelial lining leads to bleeding and activation of hemostatic mechanisms. Endothelial cells have a strong inhibitory effect on the hemostatic response, mainly through the synthesis of PGI2 and nitric oxide (NO), which are vasodilators and inhibit platelet aggregation. The synthesis of tissue factors that initiate hemostasis occurs only in endothelial cells after activation, and their natural inhibitors are also synthesized. Synthesis of prostacyclin, vWF, plasminogen activator, antithrombin, and thrombomodulin, the surface protein responsible for protein C activation, provides essential substances for platelet reactions and blood clotting.3,4

Figure 2. Vascular endothelial cells

Figure 2. Vascular endothelial cells

E. PRIMARY HEMOSTATIC RESPONSE.
The normal hemostatic response to vascular damage depends on the closely related interactions between the vessel wall, circulating platelets and platelet clotting factors. 3,4,5

1. Vasoconstriction reaction
Immediate vasoconstriction of the injured vessel and constriction of the small arteries and surrounding arterioles cause an initial slowing of blood flow to the injured area. If there is extensive damage, this vascular reaction prevents blood loss. This reduced blood flow causes contact activation of platelets and coagulation factors

Figure 3. Stages of vascular vasoconstriction at the time of injury

Figure 3. Stages of vascular vasoconstriction at the time of injury

2. Platelet adhesion
After injury to the blood vessels (endothelial lining), platelets adhere to exposed subendothelial connective tissue. Subendothelial microfibrils bind to the larger vWF multimer, which binds to the platelet membrane Ib complex. Under the influence of shear stress, platelets moving along the vascular surface GPIa/Iia (integrin 2β1) bind to collagen and stop translocation. After adhesion, the platelets become more spherical and exhibit long pseudopodia, which reinforces interactions between adjacent platelets. The Iib/IIIa receptor complex also forms a secondary binding site with vWF leading to further adhesion.

Figure 4. Platelet adhesion and aggregation5

Figure 4. Platelet adhesion and aggregation5

 

Von Willebrand factor (vWF) is involved in the adhesion of platelets to the walls of blood vessels and to other platelets (aggregation). vWF also carries factor VIII. It used to be known as factor VIII-associated antigen. vWF is encoded by a gene on chromosome 12 and synthesized by endothelial cells and megakaryocytes. The release of vWF from endothelial cells occurs under the influence of several hormones. 3,4,5

3. Platelet release reaction
Collagen exposure or the action of thrombin produced at the site of injury causes platelets to release their granular contents, which include ADP, serotonin, fibrinogen, lysosomal enzymes, -thromboglobulin, and heparin-neutralizing factor and also activate platelet prostaglandin synthesis. There is a release of diacyl glycerol (which activates protein phosphorylation via protein kinase C) and inositol triphosphate (which causes the release of intracellular calcium ions) from the membrane, leading to the formation of a labile compound thromboxane A2, which lowers cyclic Adenosine monophosphate (cAMP) levels in platelets. and trigger a release reaction. Thromboxane A2 not only strengthens platelet aggregation, but also has strong vasoconstrictive activity. Prostacyclin is a potent inhibitor of platelet aggregation and prevents platelet deposition on normal vascular endothelium

4. Platelet aggregation
ADP and thromboxane A2 are released causing more platelets to aggregate at the site of vascular injury. ADP causes platelets to swell and pushes the platelet membranes on adjacent platelets to adhere to each other simultaneously, a further release reaction occurs which releases more ADP and thromboxane A2 causing secondary platelet agglomeration. This process causes the formation of a mass of platelets large enough to occlude the area of ​​endothelial damage. The unstable primary hemostatic plug produced by this reaction of platelets in the first few minutes after injury is usually sufficient to temporarily control bleeding. It is possible that prostacyclin, which is produced by endothelial cells and smooth muscle cells in the vessel wall, plays an important role in limiting the size of the initial platelet plug.

Figure 5. Primary hemostasis2

Figure 5. Primary hemostasis2

 

5. Stabilization of the platelet plug by fibrin
Definitive hemostasis is achieved when fibrin formed by the presence of blood coagulation factors is added to the platelet mass and by platelet-induced clot retraction or compaction.

 

F. CONCLUSION

1. The hemostatic system reflects the balance between procoagulant and anticoagulant mechanisms associated with the process for fibrinolysis.
2. Platelet reactions in the form of adhesion, secretion, aggregation, and fusion as well as its procoagulant activity are very important for its function in hemostasis.
3. Endothelial cells play an active role in maintaining vascular integrity because the loss or damage to the endothelial layer causes bleeding and activates hemostasis mechanisms so that primary hemostasis begins.
4. Primary hemostasis involves blood vessels and platelets
5. Primary hemostasis is a stage of hemostasis that is rapid but does not last long.
6. The unstable primary hemostatic plug produced by this platelet reaction in the first few minutes after injury is usually sufficient to temporarily control bleeding

 

REFERENCES

  1. McMichael,M.2005.Primary Hemostasis. Journal of Veterinary Emergency and Critical Care ;15:1-8
  2. Pierce, B Tada et al. 1999.A comprehensive review of the physiology of hemostasis and antithrombotic agents. BUMC Proceedings;12:39-49
  3. Beardsley S, Diana.1990. Platelet Membrane Glycoproteins: Role in Primary Hemostasis and Component Antigens. The Yale journal of Biology and Medicine,469-475.
  4. Peran faktor von Willebrand dalam sistem hemostasis. Bagian Patologi Klinik Fakultas Kedokteran Universitas Trisakti.
  5. Hoffbrand,A.V, Petit,J.E, Moss,P.A.H. 2005. Kapita selekta Hematologi edisi 4.EGC:Jakarta.hal 221-33.
  6. Ono, A,,Westein,A,,Hsiao,S,. 2007. Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth. The American Society of Hematology ;112:90-99
  7. Setiabudy RD, Farida O. Fisiologi Hemostasis dan Fibrinolisis. Dalam Hemostasis dan Trombosis. 2012. Ed.V.FKUI. Jakarta.h.1-15.

 

By :

dr.Rini Ariani Powatu, SpA, Dr.dr.Nadirah Rasyid Ridha,M.Kes,Sp.A(K), Prof.Dr.dr.H.Dasril Daud,Sp.A(K)

PROGRAM PENDIDIKAN DOKTER SPESIALIS

DEPARTEMEN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN

UNIVERSITAS HASANUDDIN

MAKASSAR

2018

 

 

Posted in HEMATOLOGY | Tagged , , , , , , , , , , , | Leave a comment

NEUTROPHIL AND ITS CLINICAL MEANING

NEUTROPHIL AND ITS CLINICAL MEANING

A. Granulopoiesis
Leukocytes can be divided into two major groups namely phagocytes and lymphocytes. Phagocytes consist of granulocytes and monocytes. Granulocytes consist of three types of cells namely neutrophils, eosinophils and basophils. Under normal circumstances only mature phagocytic cells and lymphocytes are found in peripheral blood. The function of phagocytes and lymphocytes is to protect the body against infection and are closely related to the body’s two soluble protein systems, namely immunoglobulins and complements. (Hoffbrand, 2006)

Gambar 1. Proses hematopoiesis

Gambar 1. Proses hematopoiesis

Blood granulocytes and monocytes are formed in the bone marrow from common precursor cells. In granulopoiesis; myeloblasts, promyelocytes and myelocytes form groups of proliferative or mitotic cells. Meanwhile, metamyelocytes, stem and segment granulocytes make up the post mitotic maturation compartment. Large numbers of stem and segmental neutrophils are also stored in the bone marrow as reserves. Normal bone marrow contains more myeloid cells than erythroid cells with a ratio of 2:1 to 12:1. Under stable or normal conditions, the bone marrow storage space contains 10-15 times the number of granulocytes found in peripheral blood. After being released from the bone marrow, granulocytes take 10 hours in the circulation before moving into the tissue to perform a phagocytic function (Hoffbrand et al, 2006; Galley et al, 2006).
In the blood flow, there are two groups with the same size, namely the circulating pool which is included in the blood count and the marginal group which is not included in the blood count. Granulocytes are thought to remain in the tissue for 4-5 days before being destroyed as a regeneration mechanism. (Hoffbrand, 2006)

Gambar 2. Maturasi neutrofil

Gambar 2. Maturasi neutrofil

Granulopoiesis is influenced by intrinsic and extrinsic factors. The intrinsic factor that plays a role is the hormonal system in the body. Androgens stimulate the production of granulocytes. Extrinsic factors that affect the increase in production are endotoxin, IL-1 and TNFα. (Hoffbrand, 2006)
B. Neutrophils
Neutrophils have a diameter of 12-15 mm and a characteristically dense nucleus consisting of pale cytoplasm between 2 and 5 lobes with an irregular skeleton and containing many pink or purple granules. Granules are divided into primary granules that appear at the promyelocyte stage and secondary ones that appear at the myelocyte stage and are most abundant in mature neutrophils. Both granules are derived from lysosomes. The primary granules contain myeloperoxidase, acid phosphatase and other acid hydrolases. The secondary granules contain leachate phosphatase and lysozyme. (Hoffbrand, 2006)
Neutrophil precursors are not normally found in the peripheral blood but are present in the bone marrow. The earliest known precursors are myeloblasts, cells that are 10-20 mm in diameter, have a large nucleus with fine chromatin with 2-5 nucleoli, basophilic cytoplasm and do not contain granules. Normal bone marrow contains 4% myeloblasts. Myeloblasts with cell division into promyelocytes are larger and have primary cytoplasmic granules. These cells become promyelocytes and then become myelocytes that have specific or secondary granules. Myelocytes have denser nuclear chromatin and nucleoli are not visible. Myelocytes divide into metamyelocytes which have a grooved or horseshoe-shaped nucleus and the cytoplasm contains primary and secondary granules. (Hoffbrand, 2006).
Neutrophils are the body’s first defense mechanism if there are damaged body tissues or foreign objects enter the body. The function of these cells is closely related to the activation of antibodies (immunoglobulins) and the complement system. The interaction of these systems with neutrophils enhances the ability of these cells to carry out phagocytosis and decompose various particles. Neutrophils are able to remove ingested or phagocytosed material, and neutrophils are also able to secrete myelinperoxidase enzymes into the surrounding environment (Danes, et.al., 2002; Langdon, et.al., 2009).
The role of neutrophils in inflammation.
Inflammation is generally characterized by a vascular wall response and an inflammatory cell response. The inflammatory process ends when the causative agent is eliminated and the secreted mediators are removed. Acute inflammation occurs within minutes to days and involves fluid exudation and neutrophil migration. Chronic inflammation ensues over days to years involving lymphocytes and macrophages and leads to vascular proliferation and scar tissue formation (Mitchell et al, 2006).
Acute inflammation has three main components that contribute to clinical signs, namely:
– Vascular changes leading to increased blood flow. If there is injury, there will be changes in vascular permeability that will affect blood flow. The combination of increased hydrostatic pressure with decreased osmotic pressure results in outflow and edema. Stasis occurs due to fluid loss so that blood flow slows down. In this condition, neutrophil accumulation occurs along the endothelium (margination) and these cells begin to emigrate through the vessel wall.
– Structural changes in the microvasculature that make it easier for plasma proteins and leukocytes to leave the blood circulation to produce an inflammatory exudate.
– Emigration of leukocytes from blood vessels and accumulation at the site of injury
In inflammation, leukocytes will go to the site of injury. This series of events is called extravasation. Adhesion and transmigration of leukocytes occur through interactions between complementary adhesion molecules on leukocytes and endothelium. The stages of neutrophil adhesion and transmigration occur through the stages of endothelial activation, leukocyte rolling, integrin activation and stable adhesion and then diapedesis.
The type of leukocytes that migrate to the site of injury depends on the age of the inflammatory response and the original stimulus. In acute inflammation, neutrophils predominate for the first 6 to 24 hours and are replaced by monocytes after 24 to 48 hours. This is due to the number of neutrophils more than monocytes, faster response to chemokines and more firmly attached to certain adhesion molecules induced at the initial time. After emigration, neutrophils undergo apoptosis after 24 to 48 hours whereas monocytes live longer. (Mitchell et al, 2006)
Leukocytes having the affinity to emigrate via inter-endothelial junctions across the basement membrane and move toward the site of injury along the chemotactic agent. Exogenous bacterial products and endogenous mediators such as complement fragments, arachidonic acid metabolites and chemokines are chemotactic agents for neutrophils. (Mitchell et al, 2006)
Neutrophils produce proteolytic enzymes, toxic metabolite oxygen and acid metabolism products

m arachidonic. If produced in excess will result in local tissue damage. Under normal circumstances and in the absence of cytokines or pro-inflammatory mediators, neutrophils will undergo spontaneous apoptosis. Neutrophils will be phagocytosed by macrophages in order to prevent the release of cytotoxic to extracellular (Galley et al, 2006).
Apoptosis can be delayed if there is inflammation by proinflammatory cytokines, bacterial liposaccharides (LPS) and progranulocyte differentiation factors such as Granulocyte & Monocyte Colony Stimulating Factor (GM-CSF). Antiapoptotic protein activity is more dominant than proapoptotic and tissue damage can also delay apoptosis. (Galley et al, 2006).
Histopathological observations were carried out by counting the number of inflammatory cells (neutrophils), the formation of new blood vessels (neovascularization), the percentage of reepithelialization, and the percentage of collagen connective tissue area (Chen et al., 2005; Winarsih et al., 2010). The percentage of re-epithelialization was calculated using the following formula: length of wound with new epithelium divided by total wound length multiplied by 100%. The calculations were carried out in 10 fields of view with an objective magnification of 40 times and then averaged.

Figure 3. Neutrophil activation against infection

Figure 3. Neutrophil activation against infection

The functions of neutrophils are:
1. Chemotaxis
Mobilization and migration. Phagocytic cells will be attracted to bacteria or sites of inflammation that may occur due to chemotactic substances released by damaged tissues or by complement components. The initial process is rolling which is influenced by L-selectin and E-selectin. Then neutrophils will perform adhesion and diapedesis that is out of the vascular endothelium.
2. Phagocytosis
Recognition of foreign particles is aided by opsonization with immunoglobulins or complement because neutrophils and monocytes have receptors for the immunoglobulin Fc fragment and for C3 and other complement components.

3. Kill and digest
This method occurs in 2 ways, namely dependent and independent of oxygen. In an oxygen-dependent reaction, superoxide and hydrogen peroxidase (H2O2), are formed from oxygen and NADPH (Nicotinamide adenine dinucleotide phosphate) or NADH. In neutrophils, H2O2 reacts with myeloperoxidase and intracellular halides to kill bacteria; superoxidase (O2) can also be seen. The non-oxidative mycobactericidal mechanism requires a decrease in the pH within the phagocytic vacuole into which lysosomal enzymes are released. An additional factor is lactoferrin, an iron-binding protein present in neutrophil granules which is bacteriostatic by depleting bacterial iron.

C. Neutrophil disorders
Normal neutrophil numbers vary widely. The relative ratio of neutrophils and lymphocytes in the blood varies with age. Neutrophils increase at birth but decline rapidly in the first few days of life. During infancy these cells make up 20-30% of circulating leukocytes. The number of neutrophils in normal healthy children is 50-70% of the total circulating leukocytes or the number of ANC 1500-2500/mm3. (Behrman et al, 2007).

Absolute Neutrophil Count (ANC).
The total neutrophil value, hereinafter referred to as ANC, is the number of immature neutrophils and mature neutrophils circulating in the peripheral blood. The number of ANC generally increases when there is a bacterial infection (Howard, 2008; Levy, 2004). The ANC value can be calculated from the results of the type count by adding up the percentage of segments and stems then multiplied by the total number of leukocytes. Immature cells before stems are not included in the calculation because they are rarely present in the circulation. Normal leukocyte values ​​vary according to age and race. In the general American population, ANC < 2000/mm3 may be clinically symptomatic, whereas some individuals in Africa have ANC <1000/mm3 but are asymptomatic. (TefferI, 2005 )
A low number of ANC has an increased risk of infection. While the number of ANC more than normal indicates the occurrence of acute infection. The risk of infection and clinical symptoms that appear in accordance with the number of ANC. The lower the ANC value, the higher the risk of severe infection. (Hoffbrand, 2006)

Table 1. Distribution of the number of ANC and risk of infection (Segel et al, 2008)
Total ANC, mm3 Clinical symptoms
1000 – < 1500 Mild neutropenia
500 – < 1000 Moderate neutropenia
100 – < 500 Severe neutropenia
< 100 Profound neutropenia

Normal ANC values ​​vary with age, especially during the first week after birth. Normal leukocyte counts and ANC for children from birth to 21 years of age are shown in table 1 (Segel et al, 2008):

normal value anc

normal value anc

NEUTROPHILIA
Neutrophilia is an increase in the absolute number of neutrophils more than 8,000/mm3 in children and adults. Mechanisms that affect neutrophilia:
1. Increased production in bone marrow, by:
a. Promotes progenitor cell proliferation and final differentiation into the neutrophil series.
b. Increases mitotic activation of neutrophil precursor cells.
c. Shorten the cell cycle time in mitosis of neutrophil precursors.
2. An increase in the number of neutrophils through mobilization from the storage compartment of the bone marrow or from the marginal pool into the circulation.
3. Decreased ability of neutrophils for margination and migration to tissues.
Chronic neutrophilia results from continuous stimulation of neutrophil production or through inhibition of bone marrow feedback mechanisms. Prolonged administration of glucocorticoids and chronic inflammation are causes of chronic neutrophilia. (Behrman et al, 2007)

Factors that increase absolute neutrophil count
Neutrophil numbers can be said to increase if it exceeds 8,000. There are several factors that can increase ANC (New Health Guide, 2014), including:
1. Production increase:
a. Konal disease – myeloproliferative disorders
b. hereditary
c. reactive
2. Increased mobilization of the bone marrow pool
a. Drugs: steroids, G-CSF
b. Stress
c. Acute infection, endotoxin
d. Hypoxia
3. Reduced margin
a. Stress
b. Infection
c. Epinephrine
d. Sport
4. Decreased ability to get out of circulation
a. Leukocyte adhesion deficiency (LAD)
b. Steroids
c. Splenectomy

NEUTROPENIA
Neutropenia is a deficiency of circulating neutrophils defined as an ANC < 2,000/mm3. Neutropenia is caused due to changes in bone marrow production or excessive loss of neutrophils from the circulation. Transient neutropenia due to acquired disease lasts only a few days to weeks whereas chronic neutropenia due to immune, congenital or genetic and can last more than 6 months. (Behrman et al, 2007; Lanzkowsky, 2011)
Based on the pathomechanism, neutropenia can be caused by reduced production, impaired distribution of neutrophils in the circulation compared to the marginal pool or tissue pool (called pseudoneutropenia), increased utilization or destruction of neutrophils in the periphery, or a combination of these mechanisms.
Intravascular stimulation of neutrophils is activated by p-C5a and endotoxins can cause increased margins along the vascular endothelium, thereby reducing the number of circulating neutrophils. (Jacobson et al, 2014)
Disorders of myeloid stem cells and myeloid progenitor cells lead to decreased neutrophil production, as in aplastic anemia, acute leukemia, and myelodysplastic syndromes.
The clinical signs of neutropenia usually manifest as infection, most commonly the mucous membranes. The skin is the most common part of the infection which manifests as boils, abscesses, rashes, and prolonged wound healing. The genitalia and perirectum are also involved. However, general clinical signs of infection, such as warmth to the touch and local swelling, may be absent, as this necessitates the presence of large numbers of neutrophils. Fever in general is common and requires immediate attention in the treatment of severe neutropenia.(Smith CW, 2016)

Factors that decrease the Absolute Neutrophil Number
In general, neutropenia is classified according to its cause, namely intrinsic due to myelopoiesis defects and extrinsic due to drugs, infections, autoantibodies, and others as follows:
1. Decreased production or intrinsic defects
a. Benign ethnic neutropenia
b. Severe congenital neutropenia (SCN): sporadic (most common) or autosomal dominant, or Kostman’s disease – autosomal recessive
c. Familial benign chronic neutropenia: autosomal dominant
d. Cyclic neutropenia
e. Reticular dysgenesis
f. Pancreatic insufficiency syndrome
g. Neutropenia in metabolic diseases, eg in: glycogen storage disease (type 1B), Barth’s syndrome, idiopathic hyperglycemia, isovaleric acidemia, and thiamine-responsiveanemia in DIDMOAD syndrome (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, Deafness)
h. Neutropenia in bone marrow failure, eg in Fanconi anemia, familial congenital aplastic anemia without anomalies, and congenital dyskeratosis.
2. Increased destruction or extrinsic defects
a. Congenital
Neutropenia associated with immunodeficiency disorders, eg in XLA and dysgammaglobulinemia, abnormal cellular immunity in cartilage hair hypoplasia, IgA deficiency, Dubowitz syndrome.
b. Obtained
I. Drug-Induced
• Idiosyncratic : antiniotics (penicillins, sulfonamides), antithyroid, antipsychotics.
• Toxic suppression: cytotoxic drugs, sulfasalazine, phenitazine
• Hapten: penbicillin, propylthiouracil
II. Infection
• Viruses: HIV, EBV, Hapatitis A and B, RSV, measles, rubella, varicella, influenza
• Bacteria: typhoid, paratyphoid, tuberculosis, brucellosis.
• Rickets: ehrlichiosis
III. Bone marrow aplasia
IV. Chronic idiopathic neutropenia
V. Secondary: chemicals, radiation, immune reactions, malnutrition, copper deficiency, hypovitaminosis B12, folate deficiency
VI. Bone marrow infiltration, neoplastic: leukemia, neuroblastoma, lymphoma, rhabdomyosarcoma.
VII. Bone marrow infiltration, non-neoplastic: osteoporosis, cystinosis, Gaucher disease.
VIII. Immune
• Drug-induced: anticonvulsants
• Alloimmune (isoimmune)
• Maternofetal
• Primary: autoimmune neutropenia
• Secondary: systemic lupus erythematosus, lymphoma, leukemia, rheumatoid arthritis, HIV infection, infectious mononucleosis, autoimmune thrombocytopenia, autoimmune hemolytic anemia, Evans syndrome.
IX. Autoimmune lymphoproliferative syndrome
X. Hypersplenism

CONCLUSION
 Formation of Neutrophils: Myeloblasts Promyelocytes Myelocytes Metamyelocytes Neutrophil stems Neutrophil segments
 Decreased neutrophil count may result in an increased risk of infection.
An increase in the number of neutrophils is a marker (marker) of an infection
The function of neutrophils is divided into three phases:
1. Chemotaxis
2. Phagocytosis
3. Kill and digest

REFERENCES

  1. Behrman, R.E., Kliegman, R., Nelson, W.E. 2007.Nelson Textbook of Pediatrics. WB Saunders Company. Philadelphia.15 : 726
  2. Galley, H.F., Helmy, M., Marzouk, S., Sakka, N., Sedrak, M., Webster, N. 2006. Delayed neutrophil apoptosis in patients with multiple organ disfunction syndrome.Critical care &Shock.9 : 9-15.
  3. Guyton and Hall. 2007. Resistensi Tubuh terhadap infeksi: Leukosit, Granulosit, Sistem Makrofag-monosit, dan Inflamasi dalam :Buku Ajar Fisiologi Kedokteran. Edisi 11.Jakarta : EGC. pp: 451-459.
  4. Hoffbrand, A.V., Moss, P.A.H, Pettit, J.E. 2006.The white cells.In : Essential Haematology.5th UK : Blackwell Publisihng, pp : 94-107.
  5. Jacobson CA, Berliner N. Neutropenia. Greer JP, Arber DA, Glader B, et al, eds. Wintrobe’s Clinical Hematology. 13th ed. Philadelphia, PA: Lippincott, Williams & Wilkins; 2014. 1279-89
  6. Lanzkowsky, P. 2011. Disorders of white blood cells.In : Manual of Pediatric Hematology and Oncology. 5th USA:Elsevier. p:272-320.
  7. Mitchell, R.N., Kumar, Abbas, Fausto. 2006. Pocket Companion to Robbins & Cotran Pathologic basis of disease 7th Elsevier Inc. New York.
  8. Segel G.B et al, 2008. Neutropenia in Pediatric Practice. Pediatric Review; 29 (1) : 12-23.
  9. Smith CW. Production, Distribution, and Fate of Neutrophils. Kaushansky K, Lichtman MA, Prchal JT, et al. Williams Hematology. 9th ed. New York, NY: McGraw-Hill; 2016

By :

dr. Muh Alfian Jafar, SpA, Dr.dr.Nadirah Rasyid Ridha,M.Kes,Sp.A(K), Prof.Dr.dr.H.Dasril Daud,Sp.A(K)

PROGRAM PENDIDIKAN DOKTER SPESIALIS

DEPARTEMEN ILMU KESEHATAN ANAK

FAKULTAS KEDOKTERAN

UNIVERSITAS HASANUDDIN

MAKASSAR

2018

 

Posted in HEMATOLOGY | Tagged , , , , , , , , | Leave a comment

EXTRAVASCULAR HEMOLYSIS

EXTRAVASCULAR HEMOLYSIS

PRELIMINARY

Hemolytic anemia is anemia caused by the hemolysis process, which is the premature breakdown of erythrocytes in blood vessels. In hemolytic anemia, the lifespan of erythrocytes is shorter (normal erythrocyte lifespan is 100-120 days).

Hemolytic anemia is anemia due to hemolysis, the abnormal breakdown of red blood cells (red blood cells), either in the blood vessels (intravascular hemolysis) or elsewhere in the body (extravascular).

Hemolysis is the increased destruction of erythrocytes and causes anemia when the bone marrow cannot compensate for the loss of erythrocytes. Hemolysis is the destruction or removal of red blood cells from the circulation before their normal lifespan of 120 days. While hemolysis can be a lifelong asymptomatic condition, it most often presents as anemia when erythrocytosis cannot keep up with the rate of destruction of red blood cells. Hemolysis may also manifest as jaundice, cholelithiasis, or isolated reticulocytosis.

Based on the cause is divided into intrinsic and extrinsic. Intrinsic causes are characterized by abnormalities in erythrocytes, including membrane, hemoglobin and enzyme abnormalities. Extrinsic causes are characterized by abnormal erythrocytes accompanied by an external process that causes hemolysis. In addition, there are those who divide hemolysis based on where the destruction occurs, namely intravascular and extravascular

Intravascular hemolysis is the destruction of circulating red blood cells by releasing their contents into the plasma. Mechanical trauma from damaged endothelium, complement fixation and activation on the cell surface, and infectious agents can cause direct membrane degradation and cell damage.

Perbandingan hemolisis intravaskular dengan ekstravaskular

Perbandingan hemolisis intravaskular dengan ekstravaskular

perberdaan mekanisme hemolysis intravaskuler dan ekstravaskuler

perberdaan mekanisme hemolysis intravaskuler dan ekstravaskuler

Extravascular hemolysis
Extravascular hemolysis is more common than intravascular hemolysis. Hemolysis occurs in macrophage cells of the reticuloendothelial system (RES), especially in the spleen, liver and bone marrow because these cells contain the enzyme heme oxygenase. Hemolysis occurs due to membrane damage (eg due to antigen-antibody reactions), precipitation of hemoglobin in the cytoplasm, and decreased flexibility of erythrocytes. Splenic capillaries with a relatively small diameter and a relatively hypoxic atmosphere will provide an opportunity for the destruction of erythrocytes, possibly through a fragmentation mechanism 2,3,10

PATHOGENESIS1,2
Hemolysis is the premature destruction of red blood cells. This can lead to anemia. Extravascular hemolysis occurs in macrophage cells of the reticuloendothelial system, especially the spleen, liver and bone marrow because these cells contain the enzyme heme oxygenase. Lysis occurs when erythrocytes are damaged in both the membrane, hemoglobin and flexibility. If erythrocytes are lysed by macrophages, they will break down into globin and heme. Globin is broken down into amino acids and used as a protein synthesis material while heme is broken down into iron and protoporphyrin.
The porphyrin ring is oxidized by microsomal heme oxygenase and produces biliverdin and iron (Fe3+). Iron is released into the plasma via iron channels and binds to apotransferrin or is stored in cells as ferritin. After that, ferritin is oxidized and degraded to hemosiderin.
Iron is transported again to be stored as reserves but protoporphyrin decomposes to CO and bilirubin. Biliverdin is reduced by biliverdin reductase to unconjugated bilirubin which is water soluble and released into the plasma bound to albumin and captured by hepatocytes. Bilirubin in the blood will bind to albumin to form indirect bilirubin, which is conjugated in the liver to become direct bilirubin which is excreted in the bile thereby increasing stercobilinogen in feces and urobilinogen in urine.

Patofisiologi hemolisis ekstravaskuler

Patofisiologi hemolisis ekstravaskuler

Metabolisme Hb

Metabolisme Hb

ETIOLOGI

ETIOLOGI

Causes of extravascular hemolysis:1,2,3

Erythrocyte membrane defects:
Hereditary spherocytosis
It is an autosomal dominant disorder (75%) with defects in erythrocyte cytoskeletal membrane proteins so that they are spheroidal in shape, not easily deformed and susceptible to sequestration and destruction in the spleen. Deficiency of structural proteins bound to the erythrocyte internal membrane. Erythrocytes that lack spectrin have an unstable membrane and are easily fragmented spontaneously.

Sferosit terlihat dengan bagian tengah lebih pucat dari sekitarnya

Sferosit terlihat dengan bagian tengah lebih pucat dari sekitarnya

Hemolysis 5
a. Autoimmune hemolytic anemia
Autoimmune Hemolytic Anemia (AIHA) occurs due to the presence of autoantibodies against erythrocytes causing hemolysis by macrophages in the RES. There are two types of autoimmune hemolytic anemia, namely the warm type and the cold type. The cause of AIHA is thought to be suppression of the immune system by a virus.

1. Warm Type
Approximately 70% of AIHA cases have the warm type, where autoantibodies react optimally at 37°C. Approximately 50% of AIHA patients with warm type are accompanied by other diseases. Erythrocytes are usually coated with immunoglobulin (IgG) alone or with complement, and are therefore taken up by reticuloendothelial macrophages that have receptors for the Fc IgG fragment. Part of the coated membrane is lost so the cell becomes progressively more spherical to maintain the same volume and is eventually destroyed prematurely, especially in the spleen.
2. Cold type
Antigen antibody reactions occur at cold temperatures (< 320 C). Causes include idiopathic, infectious. Antibodies, usually IgM, are highly efficient at fixing complement, and intra and extravascular hemolysis may occur. Treatment response is not good with corticosteroid administration.

Factors that affect the site of hemolysis is the duration of the disease, namely if acute then occurs intravascular while chronic then hemolysis occurs extravascular. In addition, the type of immunoglobulin also affects the place where hemolysis occurs. When mediated by IgG, extravascular hemolysis occurs, while IgM causes extravascular hemolysis.

b. Isoimmune Hemolytic Anemia
Isoimmune hemolysis is caused by Rhesus and ABO incompatibility. Rhesus positive has rh-antigen on its erythrocytes while rhesus negative does not. Antibody formation occurs after exposure to either transfusion or pregnancy. The rhesus blood group system is the strongest antigen when compared to other blood group systems. The antigen in rhesus positive is antigen D. Anti-D is an IgG type antibody, can cross the placenta and enter the fetal circulation. New hemolytic manifestations occur in the second pregnancy due to the presence of antibodies that cross the placenta.
Hemolysis caused by ABO incompatibility occurs in infants with blood type A, B or AB with mothers with blood type O. Hemolysis occurs directly in the first pregnancy because of the presence of anti-A and anti-B which crosses the placenta.

B cells produce IgG and/or IgM which can recognize their own erythrocyte epitope

B cells produce IgG and/or IgM which can recognize their own erythrocyte epitope

Strong complement repair antibodies result in the formation of a membrane attack complex, hole punching in the red blood cell causing the red blood cell to rupture in the circulation (intravascular hemolysis). Weak complement repair antibodies produce only the C3b opsonin, and attach to the erythrocyte membrane. Immunoglobulin binding or C3b-erythrocyte binding is destroyed by macrophages (which contain receptors for the C3b and Fc portion of immunoglobulins) as they cross organs such as the spleen (extravascular hemolysis).

hemoglobin abnormalities
Sickle cell
Hemoglobin S is abnormal hemoglobin caused by the substitution of the single nucleotide base valine in place of glutamic acid at the sixth position of the beta globin chain. These changes cause changes in the structure of the erythrocyte cell membrane which causes many complications of sickle cell disease.

Figure 6. Sickle cell

Figure 6. Sickle cell

CLINICAL MANIFESTATIONCLINICAL MANIFESTATION

Clinical manifestations of hemolysis in general are the presence of jaundice, splenomegaly. In extravascular hemolysis, urine color is dark yellow due to urobilinogenuria. Anemia may also occur. If extravascular hemolysis is suspected, diagnostic tests for a specific hemolytic state based on the etiology are performed.

SUPPORTING INSPECTION1,2,3
• Routine blood
Low Hb levels with normochromic normocytic erythrocyte index can be hypochromic microcytic. The leukocyte and platelet counts were normal.
• Reticulocytosis
• Peripheral Blood smear
Peripheral blood smear examination can show morphology of erythrocytes suggesting an etiology. The most common cell types are:
– Spherocytes are found in hereditary spherocytosis, autoimmune hemolysis, hemoglobinopathies
– Eliptosis / ovalocytes in hereditary elliptocytosis
– Fragmentation (schistocytes) i.e. sharp-pointed triangular poikilocytes in microangiopathic hemolytic states
– Poikilocytosis in hemoglobinopathies

Figure 8a. Schistocytes Figure 8b. Elliptosis / ovalocyte

Figure 8a. Schistocytes Figure 8b. Elliptosis / ovalocyte

• Bilirubinemia, blood haptoglobin slightly decreased/normal
• Urobilinogenuria

Picture . Metabolism results from intravascular and extravascular hemolysis.

Picture . Metabolism results from intravascular and extravascular hemolysis.

CONCLUSION
Hemolysis is the increased destruction of erythrocytes and causes anemia when the bone marrow cannot compensate for the loss of erythrocytes. Hemolysis is based on the site of destruction, namely intravascular and extravascular.
Extravascular hemolysis is more common than intravascular hemolysis. Hemolysis occurs in macrophage cells of the reticuloendothelial system (RES), especially in the spleen, liver and bone marrow.
Causes of extravascular hemolysis:1,2,3
• Erythrocyte membrane defects:
Hereditary spherocytosis
• Hemolysis 5
a. Autoimmune hemolytic anemia
b.Isoimmunity.
• hemoglobin abnormalities
Sickle cell
Clinical manifestations of hemolysis in general are the presence of jaundice, splenomegaly. In extravascular hemolysis, urine color is dark yellow due to urobilinogenuria.

REFERENCES

  1. Arceci R, Hann I, Smith O. Pediatric hematology 3rdWiley .Blackwell publishing. 2016.p126,523
  2. Extravascular hemolysis (online), (http://ahdc.vet.cornell.edu/clinpath/modules/chem/extravasc.hem.htm)
  3. Bernadette F, Rodak, George A, Fritsma, Kathryn D. Clinical principles and applications. Elsevier Health Sciences. 2017.p 321
  4. Kenneth D. Clinical laboratory medicine. Lippincott Williams & Wilkins. 2016.p1633
  5. Maria C L. Oliveira, Benigna M. Oliveira, Murao,Zilma M V, Letícia T. Gresta, Marcos B. Viana. Clinical course of autoimmune hemolytic anemia:an observational study. J Pediatr . 2016;82(1):58-62
  6. Dhaliwal G. Hemolytic Anemia.San Francisco Veterans Affairs Medical Center/University of California–San Francisco School of Medicine, San Francisco, California Am Fam Physician.2014 Jun 1;69(11):2599-2607.
  1. Shoenfield, Y, et al (2018). Diagnostic Criteria in Autoimmune Disease. Humana Press.
  2. Bakta. Anemia Hemolitik. 2016. Available at Http//www.scribd. com
  3. Sudoyo. Anemia Hemolitik.2016. Available at Http//www.scribd. com
  1. Aljedai A. Immune Haemolytic Anaemias. 2015. Available at http/www.emedicine.com

By :

dr.Haryanty Huntoyungo,SpA, DR.dr.Nadirah Rasyid Ridha.Mkes,SpA (K), Prof.DR.dr. Dasril Daud, SpA(K)

 

Posted in HEMATOLOGY | Tagged , , , , , , , , , | Leave a comment