Bone marrow – Preparation of material and cooking preparations for microscopic examination of bone marrow

A detailed study of the cellular composition and structure of the bone marrow has begun with the introduction into clinical practice sternal puncture method, proposed M. AND. Arinkin in 1927 city, and then intravital histological Research punctate ilium using trepanobiopsy.

The method of sternal puncture

To perform sternal puncture for M. AND. Arinkin-used puncture needle biopsy, having a protective sheet, which can be set to the desired depth, depending on the thickness of skin and subcutaneous fat, ensuring puncture the back plate of the sternum.

Bone marrow nasasyvayut syringe 10-20 ml-fitting piston to provide the required vacuum. Puncture produced in the test position lying on the back at the level of attachment of III-IV ribs midline, as the front wall of the body of the sternum is thinner and has a flat or slightly concave surface, most convenient for puncture. Besides, in the field of bone marrow cells it contains a large number of.

Can ribs and punctured the spinous processes of the vertebrae (often - III and IV lumbar). When puncture of the spinous processes of the vertebrae the patient sits, leaning forward. In children, the risk of sternal puncture due to its greater elasticity, and individual differences in the thickness of the bone. Therefore children, especially newborns and infants, preferable to make a puncture in the upper third of the tibia on the inner side of the distal femoral epiphysis or calcaneus. Puncture of iliac produce 10-20 mm posterior to the anterior spine.

The method of obtaining bone marrow study by sternal puncture

The puncture site is disinfected with alcohol and an alcohol solution of iodine. Then the skin, subcutaneous fat and periosteum infiltrated fine needle 2 ml 1-2 % Novocaine. Perhaps puncturing without anesthesia or anesthesia of the skin using chloroethyl.

The syringe and puncture needle is sterilized by boiling or dry method, dried thoroughly with alcohol, then ether.

Before making a puncture, using a screw thread, a safety shield to the desired depth and the puncture inserted mandrel. Indicative data, to be followed during the installation of the safety shield at a puncture of the sternum, are shown in Table.

Directing the needle perpendicular to the sternum of its midline, rapid movement pierce the skin, subcutaneous fat and an outer plate of the sternum. At this point, the resistance decreases and the needle, as if sinking, included in the bone marrow. At the same time it is installed vertically, motionlessly. If the needle is not in a vertical position, then, without removing it, push the fuse is slightly higher and re-promoting the needle.

It should be taken into account, that cancer, multiple myeloma, osteomyelitis and other osteolytic process needle, getting into the lesion, meets less resistance and is not fixed to the bone.

After the needle has entered the bone marrow, stylet is removed and the syringe is pushed firmly. Further, pulling the plunger of the syringe, nasasyvayut no more 0,5 ml of bone marrow (when more punctate it can get a lot of peripheral blood).

If you get a bone marrow fails, then, without removing the needle, remove the syringe, reintroduced into the needle stylet and transferred to another position - above, below or to the side. Then again nasasyvayut syringe slightly punctate. After the bone marrow harvesting needle, not razъedinяя with shpricem, removed from the sternum, and the puncture site is closed sterile sticker.

The resulting material was transferred to a watchglass and quickly in the mixer nasasyvayut leukocytes or red blood cells to the mark 0,5 (under normal or elevated white blood cell count in the peripheral blood it is recommended to use a mixer-melanzhera erythrocytes, and if leukopenia - mixer melanzhera for leukocytes).

Next to the mark "11" in the mixer for leukocytes or to label "101" to the mixer for erythrocyte gaining 3-5 % acetic acid. The contents of the mixer are thoroughly mixed, discharged from the capillary part of the liquid, and subsequent drops and fill the counting chamber were counted as leukocytes blood cells, considering the dilution in a suitable mixer. Total myelokaryocytes (cells with nuclei) ranging from 50 G to 250 T in 1 l, apparently, due to unequal marrow composition in its different areas.

Counting the absolute number is not essential myelokaryocytes. In most cases, limited to counting the percentage of smear punctate various nuclear cells. At the same time the number of cells judged estimated smear.

For the preparation of drugs from the sternum punctate on a watch glass particles collected bone marrow and prepare them thin strokes, blood admixture is removed via a filter paper or a Pasteur pipette.

Of great practical importance is the proper preparation of a smear of bone marrow tissue, as an admixture of peripheral blood distorts the composition of its cells. In a properly prepared specimen cells are densely, but separately, and their structure is clearly visible. It is recommended to prepare a series of strokes, using the whole material is obtained in the puncture. Smears should be prepared quickly, since the folding of the bone marrow occurs earlier, than the peripheral blood clotting, and the cells can be damaged as, that they can not differentiate.

Aplastic bone marrow states smears revealed a small number of cells, sometimes there are no. To clarify, This is a consequence of the disease process or improperly conducted puncture, required repeated puncturing and research punctate.

Fixing and painting strokes performed in the same punctate, as well as peripheral blood smears, best of all they are colored by Pappenheim, Kryukov.

Iliac bone biopsy

The need for in vivo study of histological preparations of bone marrow, obtained by trepanobiopsii, arises, when the puncture is not possible to get enough material to confirm the presence of a pathological process.

Particularly important acquires histological methods for diseases such, As leukemia, polycythemia vera (erythremia), osteomyelofibrosis, gipoplasticheskaya anemia.

To puncture and extraction of bone tissue is proposed to use a needle-trocar, constructed on the principle of puncture-biopsy needle. The thickness of the needle-trocar 3 mm, inner diameter 2 mm, length 40 mm. The distal end of the needle like a cutter, eiloid, so that it can be cut while rotating the bone. The needle consists of a mandrel (stylet with a pointed end) and handles.

The method of obtaining bone marrow study by trepanobiopsy

Puncture produced in the iliac crest, some distance 20-30 mm posterior to the upper front spine. Technically, it is more convenient to do a biopsy of the left-wing.

The puncture site is disinfected with alcohol and an alcoholic solution of iodine. The needle is pre-sterilized by boiling or dry method, and then dried with alcohol and ether. With screw thread, a safety needle shield to the desired puncture depth, taking into account the thickness of subcutaneous fat.

Before the introduction of the trocar needle produce anesthesia of the skin, subcutaneous tissue and periosteum 2 % solution of novocaine.

Penetrating needle-trocar in the soft tissue, the lance mandrel feel for a place on the bone, where you want to do a puncture. The needle is then rotary movements under some pressure is introduced into the bone. When feeling strong fixation of the needle stylet is removed. Disconnect the mandrel and the handle, latter is screwed onto the needle, fixed in the bone. Circular movements clockwise needle is inserted into the bone. Then, as the rotational motion remove it. Cylindrical column bone, in request pins, push the stylet from the lumen of the needle on a slide, and from there transferred to a jar with formalin and sent for histological examination.

Of the remaining glass, needle and stylet of the bone marrow smears do. Most often manage to cut and remove a piece of bone in length 6 to 10 mm, and sometimes more.

In bone marrow of healthy people and patients with hyperplastic processes revealed many different cells. In severe hypoplastic (aplastic) trepanat process is yellow, due to the almost complete lack of bone marrow and replacing it with the fat tissue.

In all forms of osteomyelofibrosis myelofibrosis and extracted a piece of bone looks "dry" and from it is possible to extract only a very small amount of bone marrow for the preparation of smears.