Bone marrow

Bone marrow, in humans, is the principal hematopoietic organ; it is a soft, nutrient-rich, spongy tissue that occupies the canals of the long bones and the central fascia of the flat bones. Under normal conditions it performs a primary function in the production, maturation, and destruction of blood cells. The production of blood cells depends on the function of pluripotent stem cells. Bone marrow has two types of stem cells, mesenchymal and hematopoietic. The process of developing different blood cells from pluripotent cells is known as hematopoiesis. Pluripotent hematopoietic cells can become any cell in the blood system.

Under the influence of tissue and hormonal factors, these cells differentiate into specific blood lines. As these cells differentiate or mature, they become the cells we can recognize in the bloodstream. In addition, the bone marrow is a key component of the lymphoid system, producing the lymphocytes that form part of the body’s immune system. Having a hematopoietic function, it is divided into:

  • red bone marrow, which constitutes the main hematopoietic tissue; it is called red because, in vivo, due to the strong presence of blood and erythrocytes, it appears bright red. Bone marrow, at the beginning of fetal life, first appears in the clavicle and then spreads in all the other bones. During fetal development and for a period of several years we have mainly red bone marrow. After the first years of life, there is a progressive transformation of bone marrow in yellow bone marrow due to the loss of its hematopoietic properties, while the adipocyte and connective component increases; in the adult the active red bone marrow is localized only in some locations (some areas of the diploe of the cranial vault, sternum, ribs, vertebrae and iliac crests);
  • yellow bone marrow is present mainly in the long bones of the arms, legs and fingers. It retains its hematopoietic potential and can also become active red bone marrow by colonization of stem cells from the circulation.

In the bone marrow it is possible to recognize

  • the support tissue, formed by connective cells, the reticulocytes, which with their argirophilic fibers form a dense reticular weave that supports the cells of the hematopoietic tissue and forms a spongy reticular system that facilitates their passage and migration;
  • the hematopoietic compartment, rich in cells at various stages of maturation (erythrocytes, neutrophil granulocytes, basophil granulocytes, eosinophilic granulocytes, lymphocytes, monocytes, megakaryocytes, platelets);
  • the vascular compartment, consisting of venous sinuses or sinusoids that, in addition to having trophic function, allow the constant migration in the circulatory stream of erythrocytes and other elements of the blood. Attached to the sinusoids we find the megakaryocytes, large cells that by budding and detachment of part of the cytoplasm produce platelets.
  • It is difficult to obtain histological preparations of bone marrow because of the intrinsic delicacy of this tissue. After appropriate fixation and decalcification of bone tissue, microtome sections can be obtained, or discrete preparations can be obtained by crushing or by smears of tissue fragments.

Bone marrow examination

In the presence of one or more cytopenias, a bone marrow examination may be necessary. A bone marrow examination may reveal abnormalities in the marrow cells (e.g., dysplastic cells) and will allow evaluation of the chromosomes (cytogenetics). These tests provide additional information that may help establish the diagnosis. A bone marrow examination consists of two parts: bone aspirate and bone biopsy, both procedures are usually performed at the same time.

Bone marrow aspirate

The bone marrow aspirate is a sample of the fluid portion of the bone marrow from inside the bone cavity. The fluid is composed primarily of marrow blood and a small amount of marrow tissue. The bone marrow blood contains the hematopoietic precursors, i.e. the cells that represent the precursors of white blood cells, red blood cells and platelets, in the different stages of development from stem cells: by studying them under the microscope it is possible to understand if there are alterations as well as changes in the relationships between the different cell types. For example, in the case of an acute leukemia there will be an increase of immature cells, the so-called blasts, while in the case of a myeloma there will be many plasma cells.

The sample provides information about the shape of the cells (morphology), the maturation of the cells (differentiation) and the number of blasts (immature cells) in the bone marrow. The aspirate can also be used for other tests such as cytogenetics. This type of analysis allows for information such as:

  • myeloid/erythroid population ratio (M/E): this type of calculation provides information regarding the ratio between the number of cells in the myeloid (WBC precursors) and erythroid (RBC precursors) series;
  • differential count: determines the number of cells for each cell line (WBC, RBC and platelets), in order to determine the ratio between them;
  • Detection of the presence of abnormal cells such as leukemic or tumor cells.

Bone marrow needle aspiration therefore allows to diagnose or confirm the suspicion of the presence of cancer and, through a series of specific analysis (such as molecular biology, immunological studies, cytogenetics) to know part of its characteristics. The needle aspiration may be followed by another examination, the bone marrow biopsy.

Bone marrow biopsy

Bone marrow biopsy is an examination that consists of a special puncture, usually performed in the posterior part of the pelvis (posterior iliac crest) or alternatively in its anterior part; it is a small sample of spongy bone containing bone marrow, usually measuring about 1.5-2.0 cm. The biopsy provides information about the cellularity of the bone marrow (increased = hypercellular, reduced = hypocellular). It also provides useful information about iron deposition, fibrosis, and the presence of any other alterations.

In oncology, bone marrow biopsy is performed to diagnose tumors of the blood, the bone marrow itself or to evaluate the infiltration of tumor cells into the marrow. Specifically, the biopsy specimen is therefore used to evaluate:

  • cellularity: the volume of cells is compared with the volume of other components of the marrow, such as fat; the number of cells is evaluated to detect any increase or decrease in cells;
  • cell lines: it is evaluated if the cell lines (myeloid, erythroid and magakaryocytic) are present in adequate number;
  • the presence of abnormal cells (tumor infiltrates) or other changes in the bone marrow stroma (such as fibrosis) or bone (osteoporosis) is detected.

Depending on the suspected pathology, in addition to this test may be required other examinations that can be performed on the bone marrow sample, such as

  • tests useful to confirm the diagnosis and the staging and typing of leukemia; these tests include a particular staining or the determination of specific cell surface markers (e.g. immunophenotype performed in cytometry) that are able to provide important information about the type of leukemia present;
  • particular staining for the evaluation of iron reserves within the bone marrow and for the search of abnormal precursors of erythrocytes characterized by the accumulation of iron particles around the nucleus (the so-called ring sideroblasts);
  • chromosomal analysis and fluorescent in situ hybridization (FISH) for the detection of chromosomal abnormalities possible in leukemia, myelodysplasia, lymphoma and myeloma;
  • molecular tests as for the search of mutations useful for diagnostic and/or prognostic purposes, such as:
    • BCR-ABL1
    • JAK2
    • TCR rearrangements
    • B-cell immunoglobulin rearrangements
    • PML-RARA
  • Cell cultures for detection of viral, bacterial and fungal infections that could be the cause of unexplained febrile events. Some bacteria and fungi can also be identified with special types of staining.

Bone marrow transplantation

Bone marrow transplantation, or rather, hematopoietic stem cell transplantation is a therapy that is performed to treat congenital blood diseases (neoplastic and non-neoplastic hematologic diseases) such as thalassemias and severe immunity deficits and blood cell tumors, congenital errors of metabolism and primary immunodeficiencies. With regard to cancers such as leukemia, transplantation may be the only life-saving therapy or the most effective therapy to reduce the risk of relapse.

Bone marrow transplantation (also known as stem cell transplantation), involves the collection of hematopoietic stem cells from a healthy individual and their transfer, by transfusion, to an individual whose bone marrow is compromised and unable to produce normal blood cells. Transplantation allows the replacement of diseased cells with healthy stem cells capable of diversifying into white blood cells, red blood cells and platelets.

Types of bone marrow transplantation

Transplantation can be autologous or allogeneic. One speaks of autologous transplantation or autotransplantation when the infused hematopoietic stem cells have been taken from the patient himself in a previous phase of the disease and properly cryopreserved in liquid nitrogen at a temperature of -196 ° C. This type of transplantation is the most widely used in the treatment of solid tumors and is intended to allow high doses of anti-tumor chemotherapy. The patient can then undergo very high-dose chemotherapy which, in addition to killing the cancer cells, also kills the hematopoietic stem cells of the bone marrow. After chemotherapy, the stem cells are re-infused and differentiate into mature blood cells within a fortnight. The patient can thus face and overcome a therapy that, without autotransplantation, would have been lethal.

On the other hand, allogeneic transplantation is performed from a totally or partially compatible donor (otherwise an immune response would be triggered in the patient that would cause the destruction of the newly transfused cells, which are recognized as extreme), which can be a monozygotic twin, a sibling, a parent or a donor outside the family but present in the registry of bone marrow donors. This type of transplant is used for the treatment of malignant hemopathies, genetic diseases and in immunodeficiencies. Transplantation can be performed by different types of donors:

  • Compatible family donor (HLA-identical): this is the best option, represented by a brother (or sister) who is 100% compatible with the patient. The probability of a sibling being HLA-identical (compatible) is 25%.
  • Non-consanguineous donor: 70-75% of patients in need of a transplant do not have an HLA-identical donor within the family unit. Since the early 70’s, the establishment of International Bone Marrow Registries has made possible the access to the transplant procedure for an increasing number of subjects. It depends, however, largely on the genetic characteristics and ethnicity of the recipient. Patients of Caucasian origin have a higher probability of identifying a compatible donor than patients of African or Hispanic origin, since the ethnic groups from which these patients originate are much less present in international registries.
  • Umbilical cord: in the last two decades, numerous studies have evaluated the suitability of cord blood, collected at birth and cryopreserved in umbilical cord banks, as an alternative source of hematopoietic stem cells. Cord blood transplantation has the undoubted advantage of representing a source of hematopoietic progenitors immediately available and studies carried out in the pediatric population have shown an effectiveness comparable to transplantation from a non-consanguineous donor. In this type of transplant, the cord used can belong to either an HLA-compatible sibling or a voluntary donor.
  • Haploidentical donor (or HLA-partially compatible familial donor): despite the increasing number of potential donors available in international registries, 30-40% of patients do not find a suitable donor or are urgently in need of transplantation in times not compatible with those required to identify a donor outside the family (on average it takes 3-4 months). For this reason, in recent years much has been invested in the use as donors of hematopoietic stem cells from either parent, which are 50% HLA-compatible with their child. However, the use of these cells risks causing serious infectious or immunologic complications, potentially fatal. In order to improve the safety of parental transplantation, a stem cell manipulation technique has been developed to reduce the risks of infection and other complications that arise when using a partially compatible donor such as a parent. This technique of hematopoietic stem cell manipulation allows to eliminate the cells (alpha/beta+ T lymphocytes) responsible for the aggression of the recipient’s tissues by the donor’s cells (Graft versus host- disease or transplant versus host disease) while leaving within the transplant a high number of cells (gamma/delta+ T lymphocytes and Natural Killer cells) capable of carrying out an anti-leukemic action (in the case of malignant diseases) and of protecting the patient from serious infections, especially in the first months after transplantation.

Transplant complications

Hematopoietic stem cell transplantation has become an increasingly safe procedure over the years, thanks to improvements in tissue typing (HLA) techniques and optimization of conditioning regimens and supportive therapies. There is still, however, a non-negligible risk of complications. The main ones are:

  • rejection of transplanted cells by the recipient’s immune system;
  • Graft versus host disease (GvHD): it is a condition related to the aggression of the child’s tissues by donor lymphocytes. Distinguished are:
    • an acute form, which begins in the first 100 days and can affect the skin, intestines and liver;
    • a chronic form, which begins after the first 100 days and can take on the characteristics of a generalized autoimmune disease as it can affect different organs and tissues. In order to avoid the occurrence of this dreaded complication, a specific prevention is carried out with drugs both before transplantation and after the infusion of hematopoietic stem cells. Prevention is based on the use of immunosuppressive therapy and aims to promote a balanced coexistence between the immune system of the child and that of the donor. In patients undergoing haploidentical transplantation, as already specified, it is instead necessary to remove part of the donor lymphocytes before the infusion of stem cells;
  • infections (bacterial, viral and fungal): the risk of infections is highest in the first days after transplantation, when the patient is completely devoid of immune defenses. With the rise of white blood cells, the patient resumes to have a first line of defense against germs but several months are necessary to restore a fully functional immune system. Reconstitution of the immune system is slower in patients transplanted with hematopoietic stem cells from a haploidentical donor, because the manipulation of the transplant can delay the development of certain lymphocytes, the T lymphocytes that are essential in protecting against viral and fungal infections;
  • long-term side effects caused by chemotherapy and radiation therapy in preparation for the transplant: problems that can occur at a distance from the transplant depend on many different factors such as the type of drugs used for conditioning before the transplant, the use or not of radiation therapy or other drugs used for immunosuppression.

Problems that can occur even several years after transplantation include:

  • Damage to individual organs such as the heart, lungs, kidneys, and nervous system;
  • Tumor relapse;
  • Secondary tumors, i.e., new tumors;
  • Infertility;
  • Hormonal alterations especially to thyroid and pituitary hormones;
  • Cataracts;
  • Post-transplant lymphoproliferative diseases.
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