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Auscultation is used to study the spleen: peritoneal friction sound can be heard in perisplenitis in the region overlying the spleen.






Instrumental and Laboratory Methods

MORPHOLOGICAL STUDY OF THE BLOOD

Total blood counts are widely employed. Blood studies include quan­titative and qualitative determination of the composition of the formed blood elements: counting erythrocytes and determining their haemoglobin contents, total leucocytes and their separate forms, and platelets. Addi­tional counts are sometimes necessary depending on the character of the disease (counting reticulocytes, deriving the thrombocyte formula, etc.).

The concept of a reticular cell as a source of all cell elements of the blood has undergone a substantial revision in recent years in connection with advances in haematology. The haemopoietic scheme is now described as follows.

.The first class of polypotent precursor cells is represented by the stem cell. The stem cells are self-sustaining, characterized by rapid proliferation and differentiation.

The second class of partly determined polypotent precursor cells is represented by precur­sors of lymphopoiesis and haemopoiesis; their self-sustaining power is limited; the cells are found in the bone marrow.

The third class of unipotent precursor-cells includes colony-forming cells (precursors of granulocytes and monocytes), erythropoietin-sensitive cells, precursors of B-lymphocytes and T-lymphocytes precursors.

Thefourth class includes morphologically identifiable proliferating cells; the fifth class in­cludes maturating cells and the sixth class mature cells with a limited life cycle. The cells of the sixth class are mainly delivered to the peripheral blood.

The cell composition of the blood of a healthy individual is constant and any changes are therefore diagnostically important. But minor changes in the blood can be observed during the 24-hour period: after meals, exer­cise, etc. In order to remove these interfering factors, blood specimens should be taken under the same conditions.


Taking blood specimens. The study of blood begins with obtaining its specimen. Blood is taken from the 4th finger of the left hand. The finger is first disinfected by a mixture of alcohol and ether. The skin on the side of the first phalanx is then punctured by a blood lancet to a depth of 2.5-3 mm. Blood should issue freely because any pressure on the finger will express other tissue fluids to impair the accuracy of studies. The first emerging drop of blood should be wiped off with dry cotton wool.

Determining haemoglobin. There are the following three major groups of methods for determining haemoglobin: colorimetric (widely used in practical medicine), gasometric, and determination by the iron contained in the haemoglobin molecule. Sahli's method of estimating haemoglobin (1895) was widely used until recent times.

The cyanmethaemoglobin method has now been universally accepted as the most accurate and objective technique, which was approved by the In­ternational Standardization Committee (in haematology). The method is based on oxidation of haemoglobin (Hb) to methaemoglobin (MetHb or Hi) by potassium ferricyanide. Methaemoglobin reacts with CN-ion to form a stable red complex, cyanmethaemoglobin (CNMetHb) or haemoglobin cyanide (HiCN). Its concentration can be measured on a spectrophotometer, photoelectrocolorimeter, or haemoglobinometer.

According to this method, 0.02 ml of blood taken from the finger is transferred into 5 ml (dilution 1: 251) of a transforming solution consisting of acetone cyanhydrine, potassium ferrocyanide, and sodium hydrocar-bonate; the mixture is stirred thoroughly, allowed to stand for ten minutes, and the optical density of the solution is measured at 500-560 nm (a green optical filter) against a blank solution (the transforming solution or pure water). Concentration of haemoglobin is determined from a calibration curve. Concentration of haemoglobin in healthy people varies from 120-140 g/1 in women and from 130-160 g/1 in men.

Erythrocyte counting. In order to count erythrocytes in the chamber, blood is diluted to 1: 200 in 3.5 per cent sodium chloride solution. To that end 0.02 ml of blood is added to 4 ml of the diluting solution. The mixture is stirred thoroughly and transferred into the counting chamber.

The counting chamber is a glass plate with one or two counting grids. Biirker haemacytometers are usually used for the purpose. Three elevated strips, separated from each other by grooves pass across the main plate. The middle strip is divided into halves by another groove. Each half has a graduated counting grid. The lateral strips are 0.1 mm higher than the middle one. The cover glass rests on the elevated lateral strips to ensure a 0.1 mm spacing be­tween the grids and the cover glass (the depth of the counting chamber). In order to ensure the accurate spacing, the cover glass should be pressed tightly against the strips. A well-washed and wiped glass is ground-in by reciprocating sliding movements until the iridescent (Newto­nian) rings and lines appear over the lateral strips. A drop of diluted blood is placed by a

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Chapter 9. Diseases of the Blood



 


pipette under the ground-in cover glass. The fluid is sucked in by capillary force to fill the space over the grid.

If the blood was diluted in a test tube, the mixture should be first jolted, then a glass rod dipped into the fluid and a hanging drop transferred onto the slit between the counting chamber and the cover glass. Counting should be done one minute later (when the erythrocytes precipitate to the chamber bottom).

There exist many counting grids but they all employ one principle. They consist of larger and smaller squares with the area of 1/25 and 1/400 mm2, respectively. Goryaev's grid is commonly used in the Soviet Union. It con­sists of 225 greater squares, 25 of which are divided into smaller ones, 16 squares in each greater square. Erythrocytes are counted in 5 greater squares (divided into smaller ones). A certain rule is followed in counting: cells are counted in each square in one direction, and then this direction is reversed in the next row of squares, as shown by the arrow in Fig. 108. Counted are not only the cells inside the square but also those lying by two lines (e.g. the left and the upper line) without counting blood cells lying on the right and lower line. The quantity of erythrocytes counted in 5 greater squares is recalculated with reference to one litre.

Normal erythrocyte counts in women are 3.9-4.7 x 1012 and in men 4-5 x 1012per 1 1 of blood.

OH-

There are instruments by which the counting procedure is either simplified or automated. These are erythrohaemometres and absorp-tiometres where concentration of erythrocytes is assessed by the amount of absorbed or scattered light passed through a suspension of erythrocytes, or directly reading automatic instruments. In the latter case blood cells pass a narrow capillary to change resistance of an electric circuit. Each cell gives a

Fig. 108. Counting red blood cells.


pulse on the screen of an oscilloscope and is recorded on the instrument scale.

Once the quantity of erythrocytes and haemoglobin in a given blood specimen is known, it is possible to calculate the haemoglobin content of each erythrocyte. There are many methods by which haemoglobin satura­tion can be determined. One of them is the calculation of the colour index. This is a conventional value derived from the ratio of haemoglobin to the number of erythrocytes. This value is found by dividing a tripled quantity of haemoglobin in grams by the first three figures expressing the quantity of erythrocytes. Normally this value approaches 1. If it is less than 1, the erythrocyte saturation of haemoglobin is insufficient; if the value exceeds 1, the volume of erythrocytes is higher than normal. Oversaturation with haemoglobin is impossible. A normal erythrocyte is saturated with haemoglobin to the utmost limit.

At the present time, in accordance with the general tendency to express blood constants in absolute values, the weight percentage of haemoglobin in erythrocytes is calculated, instead of determining the colour index of the blood. To that end haemoglobin content in one litre is determined and the found quantity divided by the number of erythrocytes in the same volume. Normally one erythrocyte contains 33 ng of haemoglobin.

Leucocyte counting. Blood for counting leucocytes is diluted either in a special mixer or a test tube. A 3-5 per cent solution of acetic acid destroy­ing erythrocytes is mixed with a small amount of a suitable aniline dye to stain leucocyte nuclei. The counting chamber is filled as for counting erythrocytes. It is convenient to count leucocytes in 100 greater (undivided) squares. A constant factor is found from the dilution of blood and the volume of fluid in each square. With 1: 20 dilution it is 50. When test tubes are used for dilution, 0.02 ml of blood is added to 0.38 ml of the diluting liquid in the test tube. Saponin is used for haemolysis of erythrocytes in automatic counting instruments. The normal leucocyte counts are 4000-9000 in 1 jtl or 4.0-9.0 X 109 per 1 1 of blood.

The leucocyte formula is counted in stained smears. An adequate smear meets the following requirements: it is thin and the formed elements are ar­ranged in one layer; the smear is yellow and semitranslucent. The width of the smear is 2-3 mm narrower than the glass, while the length, 2/3-3/4 the length of the glass. A good smear is uniform and the cells are intact (not damaged during their application to the glass). In order to ensure an even layer, the glass is first defatted over a gas burner or in a mixture of alcohol and ether. A small drop of fresh blood is touched by the glass edge and spread immediately over the entire glass surface. A polished cover glass of the counting chamber or another object glass with polished edges and made slightly narrower than the main object glass can be used for the purpose.

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Chapter 9. Diseases of the Blood



 


This glass is positioned behind the blood drop at an angle of 45° to the plane of the first glass and moved back to bring it in contact with the blood. As soon as the blood spreads over the entire width of the polished edge, the glass is moved forward along the surface of the object glass. Blood is thus spread in an even layer over the object glass. Before staining, the smear is fixed in methanol for 3 minutes, or in ethanol or a mixture of ethanol and ether for 30 minutes. Other fixing agents can also be used. When the smear is dry it is covered with a layer of stain.

Differential staining is used for blood cells. Romanovsky-Giemsa staining method is com­monly used. The stain is a mixture of weakly acid (eosin) and weakly alkaline (azure II) stains. Depending on the reaction of the medium, the cells and their parts differently accept the stain: acid (basophilic) substances are coloured blue by azure, while alkaline (oxyphilic) substances are coloured red by eosin. Neutral substances accept both dyes and turn violet. Azure II, which is generally blue, contains a small quantity of azure I. In some cells the cytoplasm con­tains grains which selectively accept red azure I. The grains are called azurophilic.

Romanovsky-Giemsa stain is diluted before use with distilled water, 1-2 drops per 1 ml of water. Smears are placed on glass rods fixed in the sides of the cell and the stain is added in the maximum quantity that can remain on the glass. The staining time (15-30 min) depends on concentration of the stain, quality of water (neutral) and temperature; it is determined em­pirically. The stain is then removed by a jet of water and the smears are placed in the vertical position to dry.

Differential blood count is the percentage of separate forms of blood leucocytes. In order to ensure accuracy, it is necessary to observe not less than 200 leucocytes using the immersion system. Since the cells are not evenly distributed over the surface (larger cells tend to move toward the edges) it is necessary to follow a certain rule in counting, so that both the centre and the peripheral parts of the smear might be inspected. The smear can be moved from its upper edge to the lower one, then in the lateral direc­tion, through 2 or 3 fields of vision, then back, from the lower to the upper edge, and so on. According to another method, the smear is moved from the edge, through 5 or 6 vision fields toward the centre, then the smear is moved in the lateral direction through the same distance, then again to the periphery, and so on, until 50 cells are counted. Four sites by the four angles of the smear should be thus inspected. Each cell should be identified and recorded. A special 11-key counter is convenient for cell counting. When 200 cells are thus counted, the number of each leucocyte is divided by two.

Leucocytes quickly respond to various environmental factors and changes inside the body. Shifts in their counts are very important diagnostically. But individual variations in leucocyte composition are quite significant and it is therefore necessary to compare individual findings not with the average values, but with a certain range within which these varia­tions are normal (see Appendix, Table 6).


When assessing the composition of leucocytes, it is necessary to bear in mind that changes in percentage ratios can give an incorrect picture of the shifts occurring in the blood. For example, an increase in the absolute amount of a given type of cells in the blood decreases the percentage of all other cell elements. The picture is reverse with decreasing absolute amount of this given type of blood cells. A correct conclusion can be derived not from relative (percentage) but absolute values, i.e. the quantity of a given type of cells contained in 1 fil (in 1 1 of blood, according to the SI).

The total quantity of leucocytes alone is of great diagnostic significance, because it characterizes the condition of the haemopoietic system and its response to harmful effects. The increased number of leucocytes (leucocytosis) is the result of activation of leucopoiesis. The decreased number of leucocytes (leucopenia) depends on the inhibition of the haemopoietic organs, their exhaustion, increased decomposition of leucocytes under the effect of antileucocytic antibodies, etc.

Neutrophils are the most changeable group of leucocytes. Their number increases in many infections, intoxication, and tissue decomposition. Neutropoiesis is characterized not only by the increased total number of neutrophils but also by the appearance in the blood of immature forms: the quantity of stab neutrophils increases; juvenile neutrophils and even myelocytes appear. This rejuvenation of the neutrophil composition is call­ed the blood shift to the left, because the figures grow on the left side of the laboratory blank where leucocyte counts are normally recorded. Regenerative and degenerative shifts are distinguished. In the regenerative shift to the left the mentioned changes are observed, while in the degenerative shift to the left, the number of stab neutrophils only increases along with the degenerative changes in neutrophils in the absence of leucocytosis (vacuolization of cytoplasm, nuclear pyknosis, etc.). The regenerative shift indicates active protective response of the body, while the degenerative one indicates the absence of this response. The protective role of neutrophils consists in phagocytosis, bactericidal action, and production of proteolytic enzymes promoting resolution of necrotized tissue and heal­ing of wounds.

The regenerative shift to the left occurs most frequently in the presence of an inflammatory or necrotic focus. An especially marked shift to the left (to promyelocytes and even myeloblasts in the presence of significant leucocytosis) is called leucaemoid reaction. The number of neutrophils decreases (absolute neutropenia) in the presence of the inhibiting action of toxins of some microbes (e.g. causative agents of typhoud fever or brucellosis) and viruses, ionizing radiation, and some medicinal prepara­tions.

The absolute number of lymphocytes increases less frequently. Lym-



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phocytosis occurs during recovery in acute infectious diseases, infectious mononucleosis, infectious lymphocytosis, lymphoid leucosis, rubella brucellosis, and thyrotoxicosis. More frequently lymphocytosis is only relative, associated with a decreased number of neutrophils (like relative lymphopenia in the presence of increased number of neutrophils). Ab­solute lymphopenia occurs in radiation sickness and systemic affections of the lymphatic system: lymphogranulomatosis and lymphosarcoma.

Eosinophils are present in the blood in relatively small quantity but their number increases, and sometimes significantly, in allergic processes (serum sickness or bronchial asthma), in helminthiasis, and itching der-matosis. Eosinophilia in allergic processes is associated with the role played by eosinophils in removal of toxic substances produced in these reactions. Decreased number of eosinophils (eosinopenia), to their complete absence, occurs in sepsis, severe forms of tuberculosis, typhus, and poisoning.

Basophils are carriers of important mediators of tissue metabolism. Their number increases in sensitization of patients and decreases markedly during decomposition caused by the repeated administration of the allergen.

Increased number of monocytes (monocytosis) indicates development of the immune processes. Monocytosis occurs in some chronic diseases (e.g. chroniosepsis, tuberculosis, malaria, visceral leishmaniasis, syphilis) and in infectious mononucleosis. Monocytopenia sometimes occurs in severe septic (hypertoxic) forms of typhoid fever and other infections.

Leucocyte counting procedure requires special skill. The laboratory technician should be able to differentiate between various blood cells (Plate 27). Granulocytes have specific segmented nuclei (violet like in all leucocytes) and oxyphilic (pink) cytoplasm containing grains. Grains of a neutrophilic leucocyte (10-15 ixxa) are small, their size varies; they are stained brown-violet. The nucleus has a rough structure, with alternation of intense- and light-coloured sites; it consists of 2 to 5 (mostly 3 or 4) segments of various size and shape connected by thready bridges. The nucleus of a stab neutrophil is about the same size and colour, but it is a uniform curved band which never thins to a thread. The eosinophil nuclei consist mostly of two symmetrically arranged segments of about the same size (three segments can also be present); their structure and colour are similar to those of neutrophil segments. Eosinophils are highly granular. Grains are large, round, bright-orange and of equal size; they stuff the en­tire cytoplasm. The diameter of the cell is about 15 |im. A basophil is slightly smaller than the other granulocytes (9-14 /xm). The nucleus can be segmented. Often it has an irregular oval shape and is stained intensely. The grains are large and dark-violet; their size varies. Due to metachromasia, their dark-blue colour makes them look violet.


Agranulocytes are characterized by a non-segmented nucleus and basophilic (blue) cytoplasm. The lymphocyte is the smallest of all leucocytes; its diameter usually varies between 7 and 12 /*m, but some lym­phocytes are as large as 12-15 /im. The nucleus is round, oval, or bean-shaped; it occupies almost the entire cell and is intensely coloured. The cytoplasm of most lymphocytes surrounds the nucleus by a narrow circle; it is pale-blue and becomes lighter toward the nucleus. In addition to these " small" lymphocytes, there are " medium size" ones having a large sky-blue zone of a cytoplasm. Some lymphocytes have several large cherry-red (azurophilic) grains in their cytoplasm. A monocyte is the largest blood cell. Its diameter is 20 ixm. Its large nucleus is of irregular shape and relatively light-coloured. The cytoplasm is greyish-blue and smoky; the col­our intensity does not diminish toward the nucleus. If stained well, dust-like azurophilic granularity is revealed in some cells.

In rare cases, apart from the mentioned cells, normal blood contains plasma cells. Their number increases in pathology. The cells have an eccen­trically arranged dense nucleus (often a wheel-like structure) and a marked­ly basophilic vacuolized cytoplasm. Their number increases in certain in­fectious diseases, wound sepsis, hypernephroma, myeloma, etc. These cells are probably responsible for the production of gamma globulins.

When counting leucocytes, it is necessary to pay attention to both quan­titative and qualitative shifts in the formed elements. The degenerative shifts were discussed above. In grave toxicosis, granularity of neutrophils becomes even more pronounced, the granules become larger and coloured; this granulation is called toxicogenic. Indistinct spots are sometimes reveal­ed in blood smears; they are stained like the nuclear substance of leucocytes. These are Botkin-Gumprecht shadows, the remains of nuclear chromatin characterizing brittleness of leucocytes due to which they decompose (leucocytolysis).

Erythrocytes are studied in the same smears (Plate 28). The size, shape, colour and cell inclusions should be assessed. Normal erythrocytes in the smear are rounded, their diameter varying from 6 to 8 fim (the average diameter, 7.2 jtm). The size of erythrocytes often changes in anaemia of various nature. Various erythrocytes change differently. Excessive varia­tion in the size of erythrocytes is called anisocytosis. Prevalence of smaller erythrocytes (microcytosis) occurs in iron deficiency anaemia. Macrocytosis develops in haemopoietic dysfunction of the liver. Megalocytes (large, over 12 |im, oval hyperchromic erythrocytes formed during maturation of megaloblasts) appear in the blood of patients with vitamin B12 deficiency (vitamin B12 deficiency anaemia). In pathological conditions of erythrocyte maturation, along with anisocytosis, the change in the shape of erythrocytes {poikilocytosis) is also observed; in addition to



Special Part


Chapter 9. Diseases of the Blood



 


round erythrocytes, blood contains also erythrocytes of oval, pear-shaped and other configurations. If erythrocytes are undersaturated with haemoglobin (colour index less than 0.85) they are poorly stained to become hypochromic; in vitamin B12 deficiency they are coloured intense­ly, i.e. hyperchromic (colour index higher than 1). A mature erythrocyte is oxyphilic, i.e. coloured pink. An immature erythrocyte is polychromatophilic. In supravital staining these erythrocytes appear as reticulocytes (see below). Normal blood contains polychromatophilic erythrocytes in meagre quantity: single cells per 1000 erythrocytes. Since they are less noticeable than reticulocytes, the latter are counted to assess the number of juvenile polychromatophilic cells. The importance of this count is that the number of reticulocytes in the blood is a measure of the activity of the bone marrow. Normally this number is 2-10 per 1000 erythrocytes. Erythropoiesis is activated in blood loss and haemolysis, and the number of reticulocytes in normal bone marrow and peripheral blood increases. The absence of this increase indicates decreased function of the bone marrow and conversely reticulocytosis in the absence of anaemia in­dicates latent but well compensated loss of blood. High reticulocytosis is observed in effective treatment of vitamin B12 deficiency anaemia.

In erythropoietic hypofunction of the bone marrow, more immature nuclear (but still containing nuclei) elements of the red blood, i.e nor-moblasts and erythroblasts, are delivered into the blood from the bone marrow. During maturation of erythrocytes in pathological conditions, nuclear remnants, known as Jolly bodies, may be preserved. These are round chromatin formations 1-2 /*m in size, stained cherry-red. Red Cabot rings (thread-like rings or convolutions) may also remain. They are believed to be the remnants of the nuclear envelopes, and occur mostly in vitamin B12 deficiency anaemia.

Basophilic granulation of erythrocytes is also the result of their abnor­mal maturation. Blue granules are seen against the pink background during ordinary staining of a fixed smear. It should not be mistaken for reticulocyte granulation which is revealed only in supravital staining. Basophil-granular erythrocytes occur in pernicious anaemia and some in­toxications, especially in lead poisoning.

Reticulocytes are stained in unfixed smears of fresh blood in which erythrocytes are still alive. Various alkaline dyes are used to stain smears by various techniques. Best results are attained with brilliant cresyl blue. A drop of a saturated alcoholic solution of the stain is applied to a defatted object glass and a smear is made by the usual way. As soon as the stain dries up, a thin blood film is smeared over it and the glass is transferred to a moist chamber (a Petri dish containing a piece of wet blotting paper). The smear is kept there for 5 minutes and then removed and allowed to dry.


The smear is inspected with an immersion system. Mature erythrocytes are stained green. Against this background, reticulocytes (depending on their maturity) have blue granules, filaments, or other formations that may resemble a crown, a ball, or a network. Filaments and grains are more mature forms and they usually predominate in reticulocytes.

When counting reticulocytes, their number per 1000 erythrocytes is determined. For convenience of counting, the vision field of the microscope is diminished by placing a special window in the eye-piece. The total number of erythrocytes and reticulocytes is counted in the field of vi­sion. Counting is continued till the number of erythrocytes is 1000.

Thrombocytes (platelets) have a diameter of 1.5—2.5 /im. Their normal number is 180.0-320.0 x 109 per 11 (180 000-320 000 per 1 pi) of blood. Using the Romanovsky-Giemsa staining technique, the central part, the granulomere with intense azurophilic granulation, and non-granular hyalomere around it are distinguished. If the number of thrombocytes decreases significantly (thrombocytopenia), a tendency to haemorrhages develops. The critical figure at which haemorrhage occurs is believed to be 30 x 109 per 11 (30 000 per 1 /xl). Thrombocytopenia occurs in affection of the bone marrow by infectious causative agents, some medicinal prepara­tions, ionizing radiation, and in auto-immune processes. Thrombocytosis occurs after haemorrhage, in polycythaemia, and malignant tumours.

In order to determine the number of thrombocytes, it is necessary to prevent their agglutination. To that end, a drop of a 14 per cent magnesium sulphate solution is placed over the puncture point on the finger. The blood issuing from the wound mixes with the solution and smears are made from this mixture, which are then fixed and stained after Romanovsky-Giemsa. The fixing and staining time should be doubled (compared with the blood smear staining time). Using a window to restrict the field of vision (like in counting reticulocytes), 1000 erythrocytes and all thrombocytes that occur among them are counted in vision fields. Once the number of erythrocytes in 1, ul is known, the number of thrombocytes can be calculated in 1 /xl and in 1 1 of blood.

Apart from the described indirect counting of thrombocytes, they can also be determined directly in a counting chamber. The blood is diluted by a suitable solvent, e.g. by a 1 per cent ammonium oxalate solution. A phase contrast microscope is used for counting. This method is more accurate than indirect counting. In certain diseases of the haemopoietic organs, thrombocyte counts are also necessary. Juvenile, mature, and old throm­bocytes are distinguished. They also differ in size, shape, colour and struc­ture; their degenerative forms appear sometimes.

Changes in the morphological composition of the blood should be used



Special Part


Chapter 9. Diseases of the Blood



 


to establish diagnosis of a disease together with the other findings of ex­amination of the patient.

Erythrocyte sedimentation rate (ESR). Erythrocytes do not clog together in the stream of blood because they are all negatively charged. If a blood specimen is placed in a vertical vessel and an anticoagulating agent is added to it, erythrocytes gradually settle by gravity. Then they agglomerate into heavier groups which precipitate at a faster rate. Agglomeration is promoted by some protein components of the plasma (globulins, fibrinogen) and by mucopolysaccharides. Therefore, the processes which increase their accumulation in the blood are attended by acceleration of erythrocyte sedimentation. This condition occurs in most inflammatory processes, infections, malignant tumours, collagenoses, nephroses, and tissue decomposition; to a certain measure, this acceleration is propor­tional to the gravity of the affection. In certain diseases erythrocyte sedimentation is not accelerated in their initial stage (epidemic hepatitis, typhoid fever); in other pathological conditions erythrocyte sedimentation rate is slowed (heart failure).

Erythrocyte sedimentation rate is not an independent diagnostic symp­tom; it only indicates the activity of the process. It is important in this aspect in the diagnosis of tuberculosis, rheumatism, and collagenosis. Changes in the erythrocyte sedimentation rate do not always agree with other signs of activity. For example, ESR lags behind the rate of temperature elevation and leucocytosis in appendicitis or myocardial in­farction; its normalization is also slower than normalization of the men­tioned symptoms. The normal ESR does not rule out the presence of disease which would be usually attended by an increased erythrocyte sedimentation rate. But it should be remembered that ESR does not in­crease in healthy people.

The Panchenkov method of ESR determination is widely used in the Soviet Union. A Panchenkov capillary graduated in 1 mm (100 divisions) is used for the purpose. It is filled with a 5 per cent sodium citrate solution to a half of full capacity (50 divisions). The solution is blown out onto a watch glass or into a test tube. Using the same capillary, 100 mm of blood is taken from the punctured finger (2 times). To that end, the capillary is held horizontally and brought in contact with the issuing drop of blood: the blood is drawn in by the capillary force. The blood is then mixed with the reagent in the 4: 1 ratio. The mixture is taken into the capillary, to the mark 0 (100 divisions), and placed in a Panchenkov stand, in a strictly ver­tical position. The number of millimetres of a settled plasma column is noted in 60 minutes. The normal rate for men is 2—lOmm/h and for women 2—15 mm/h.


PUNCTURE OF HAEMOPOIETIC ORGANS

The morphological composition of the blood does not always show the changes occurring in the haemopoietic organs. For example, the cell com­position of blood remains almost unaltered in aleukaemic form of leucosis despite significant changes in the bone marrow. M. Arinkin (1928) pro­posed asternal puncture for intravital study of the bone marrow. Owing to the simplicity and safety of the procedure, it is used for the study of almost all patients with diseases of the haemopoietic system. The Kassirsky needle is used for the purpose in the Soviet Union. This is a short thick-walled needle with a mandrin and a stopping device that prevents deep penetration of the needle. After the skin, subcutaneous fat and the periosteum are anaesthetized, the soft tissues are punctured over the sternum, at the level of the second or third intercostal space (or above the manubrium). Then the stopping device is fixed at a distance of 5 mm from the skin surface and the anterior plate of the sternum is punctured: the operator's hand has a feeling of entering a cavity. The mandrin is now removed and a dry 10-20 ml syringe is attached to the needle. About 0.5 or 1 ml of bone mar­row is now aspired into the syringe and transferred onto a watch glass. If the bone marrow is mixed with an unknown quantity of blood, its com­position cannot be definitely determined. Using a blotting paper (or by in­clining slightly the watch glass), the blood is separated, and the small grains of bone marrow are carefully pressed against the glass to prepare a smear of the crushed marrow. After fixation and staining (Romanovsky-Giemsa), not less than 500 elements containing nuclei are counted in the smear. A myelogram is then derived (see Appendix, Table 13).

The marrow specimen can show upset maturation of the cells: increased number of juvenile forms or prevalence of primary undifferentiated elements, upset proportion between the red and white cells, changes in the total number of cells, presence of the pathological forms, etc. Apart from the sternum, other bones (e.g. iliac bone) can also be used for taking the bone marrow.

More accurate information on the composition of the bone marrow is given by trepanobiopsy. A special needle (troacar) is passed into the iliac crest to cut out a column consisting of the bone-marrow tissue, which is then used for making histological preparations. The structure of the bone marrow remains unchanged in the preparations while the absence of blood makes it possible to evaluate its cells composition and to reveal focal and diffuse changes in it.

Enlarged lymph nodes are often punctured. It makes it possible to establish the character of changes in the cell composition and to verify the diagnosis of some systemic diseases of the lymph apparatus (lymphoid



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Chapter 9. Diseases of the Blood



 


leucosis, lymphogranulomatosis, lymphosarcomatosis), to reveal me-tastases of tumours, etc. More accurate data can be obtained with biopsy of the lymph node. The puncture is made without anaesthesia, by a simple injection needle attached to a 10-ml syringe. The obtained material is used to prepare smears. The spleen is punctured by the same method. The pa­tient is asked to keep breath at the inspiration height to prevent possible in­jury of the spleen during respiratory movements. Combined study of cell composition of the bone marrow, spleen and lymph nodes reveals the rela­tions between these organs of the haemopoietic system and the presence of extramedullar haemopoiesis which develops in some affections of the bone marrow.

ASSESSMENT OF HAEMOLYSIS

Evaluation of haemolysis becomes necessary mainly in anaemia of the haemolytic character. Erythrocytes undergo constant decomposition in physiological conditions (haemolysis). In pathological haemolysis, haemoglobin destruction is intensified to increase formation of unbound bilirubin and excretion of stercobilin with faeces and urine. This is an im­portant symptom of pathological haemolysis (see " Liver and Bile Ducts").

Another sign suggesting haemolysis is the degree of osmotic stability (resistance) of erythrocytes. Congenital microspherocytic haemolytic anaemia is characterized by decreased osmotic stability of erythrocytes. This anaemia is diagnosed by mixing blood specimens with sodium chloride solutions whose concentration increases in 0.02 per cent gradient from 0.2 to 0.7 per cent (1 ml of each solution). The mixtures are shaken and the test tubes are allowed to stand for 5-20 hours to complete sedimentation of erythrocytes (or the liquids are centrifuged after 1-hour standing). The test tubes where haemolysis takes place are separated. The minimum resistance is determined by the test tube where the concentration of sodium chloride is the highest and the pink colour becomes appreciable. The maximum resistance is determined by the test tube where the concen­tration of sodium chloride is the lowest and in which there is no sediment. Normally haemolysis begins at sodium chloride concentrations from 0.42 to 0.46 per cent and terminates at 0.30 to 0.36 per cent. In haemolytic anaemia haemolysis begins at 0.54-0.70 per cent and ends at 0.40-0.44 per cent concentration of sodium chloride.

The third sign of haemolysis (also only relative) is reticulocytosis. In­creased decomposition of erythrocytes stimulates erythropoiesis. The number of reticulocytes increases although the increase is not always pro­portional to the degree of haemolysis.


STUDY OF THE HAEMORRHAGIC SYNDROME

Blood in the human body is liquid because of the physiological dynamic equilibrium of the coagulation and anticoagulation systems. If the activity of any procoagulant decreases or is lost, or the activity of anticoagulants increases, there develops a tendency to haemorrhage (haemorrhagic diathesis). If the relation is reversed, the tendency develops to increased coagulability of the blood and formation of thrombus. Bleeding in haemorrhagic diathesis is associated with haemorrhage of fine capillaries, while haemostasis is effected by a series of sequential mechanisms which protect the body from profuse loss of blood.

The first event leading to haemostasis is formation of a white thrombus consisting of thrombocytes which have undergone the so-called viscous metamorphosis. This term is used to describe a series of consecutive phases in the transformation of the thrombocyte: after a blood vessel is injured, thrombocytes stick to the injured site (adherence) and fuse (aggrega­tion). Blood platelets stick together to lose their usual shapes and to turn into a clot that ar­rests bleeding from the injured capillary or a larger vessel before a red thrombus is formed. The platelets then dissolve to liberate substances promoting coagulation of blood, contraction of the vessel (serotonin), and consolidation of the clot. The event following formation of the white thrombus is activation of plasmic, tissue, and thrombocytic factors which cause precipitation of fibrin threads, coagulation of blood, and formation of a red thrombus, which is larger and stronger than the white thrombus.

Coagulation of blood is a complicated enzymatic process, in which 13 plasma factors (I-XIII) and 12 thrombocytic factors (1-12) are involved. The plasma factors of blood coagulation are as follows: I-fibrinogen-fibrin, II-prothrombin-thrombin, III-thrombo-plastin, IV-Ca ions, V-proaccelerin, Vl-accelerin, VH-proconvertin, VUI-antihaemophilic globulin, IX-plasma thromboplastin component, X-Koller factor, Xl-plasma thromboplastin antecedent, XH-contact factor, and Xlll-fibrinase (fibrin-stabilizing factor). According to the activation sequence (" the cascade theory"), each plasma factor of the coagulation system is a proenzyme which is activated by the preceding factor and, in turn, activates its successor, thus to ensure a kind of a chain reaction.

The blood coagulation process can be divided into three phases. The first begins at the mo­ment when the blood contacts the rough surface of the injured vessel to activate the first link in the chain (contact factor, XII) and to complete formation of thromboplastin (factor III). Thromboplastin is formed from the antihaemophilic globulin of plasma (VIII) with participa­tion of plasma factors XII, XI, X, IX, V, and three platelet factors in the presence of the calcium ions.

The second phase of blood coagulation begins with formation of thromboplastin: the blood prothrombin (produced by the liver with involvement of vitamin K) is activated by thromboplastin in the presence of the calcium ions, plasma factors VII and VI, and the I thrombocytic factor to convert into an active thrombin. Thrombin acts on the fibrinogen of blood to form fibrin. This is the third phase which ends by formation of a blood clot, i.e. the red thrombus. The next stage is the action of the fibrin-stabilizing factor of the fibrin. Under the action of the 6th platelet factor, retractozyme, fibrin threads shorten to contract and con­solidate the clot, which accounts for a complete discontinuation of the bleeding.

In addition to the factors promoting coagulation, the blood contains also anticoagulants or inhibitors of blood coagulation which are responsible for the liquid state of normal blood. Each component of the coagulation system has its opponent inhibitor (antithromboplastin,



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Chapter 9. Diseases of the Blood



 


antithrombin, anticonvertin, etc.). There are inhibitors to anticoagulants too. In physiological conditions, a change in any factor causes a corresponding change in its antagonist; the equilibrium of the two systems is thus maintained. Imbalanced increase in anticoagulant ac­tivity results in bleeding. Heparin is the most powerful anticoagulant. It inhibits all phases of blood coagulation, especially conversion of prothrombin into thrombin. Thrombocytic fac­tors play an important role in the described processes. Some of them promote coagulation of blood, and others activate anticoagulants.

After the blood clot fulfils its purpose, the reverse process is started, its dissolution. It is attained through the action of a complicated enzymatic fibrinolytic system, which in many respects is similar to the coagulation system. Fibrin of the clot is dissolved by the proteolytic enzyme fibrinolysin which circulates in the blood as an inactive profibrinolysin. It is activated by fibrinokinase (plasmic, tissue, and bacterial). There are the corresponding inhibitors of fibrinolysin and fibrinokinase: antifibrinolysin and antifibrinokinase.

It is clear that haemostasis is a very complicated phenomenon and it is sometimes difficult to find the defective link in this chain of processes. There are many tests that can reveal predisposition to bleeding or thrombus formation and to find their causes. Classical tests are distinguished by which the general coagulation trends of a given blood can be determined and which are used to examine all patients with haemorrhagic diathesis. There are also differential tests by which a missing factor can be found. The classical tests are used to determine (1) blood coagulation time; 2) thrombocyte count; (3) bleeding time; (4) retraction of blood clot; and (5) permeability of capillaries.

Coagulation time characterizes coagulability of blood in general without accounting for separate phases of the coagulation process. Coagulation time increases in increased anticoagulation activity of blood or decreased concentration of procoagulants and shortens in the presence of the tendency to thrombus formation. The longest coagulation time (to several hours) is observed in haemophilia A. It does not change in certain haemorrhagic diatheses.

In order to evaluate coagulability of blood, a venous blood specimen is placed in a test tube and kept on a water bath at a temperature of 37 °C. At 30-second intervals, the test tube is inclined and inspected to see if the li­quid level is mobile. In physiological conditions, the blood coagulates in 5-10 minutes (Lee and White method).

Drop tests are widely used to determine coagulability of blood. A specimen of blood is taken either in a capillary pipette and the time when it loses mobility is determined, or a drop is placed into a moist heated chamber onto a paraffin-coated watch glass and the time, when the drop does not flow toward the edge of the inclined glass, is determined.

Estimation of bleeding time (by Duke's method). The finger tip or an ear lobe is punctured by Franke's needle or a blood lancet to a depth of 3 mm. The spontaneously issuing blood is removed at 30-second intervals by touching it with blotting paper. The normal bleeding time is 2-4


minutes. Since discontinuation of bleeding is associated with formation of a white thrombus, the test results depend on the number of thrombocytes and the ability of the vascular wall to contract, which is promoted by liberation of the vasoconstricting factor, serotonin, by thrombocytes. The bleeding time in trombocytopenia is considerably prolonged and the number of blood drops removed by the blotting paper increases many times (Plate 29, a and b). If the capillary tone is abnormal the size of blood drops increases.

Clot retraction also depends on the number and activity of throm­bocytes since it occurs under the effect of retractozyme liberated by the blood platelets. A specimen of venous blood (3-5 ml) is placed in a graduated centrifuge test tube and placed in a thermostat at a temperature of 37 °C. The serum separated in 24 hours is removed and its volume is divided by the volume of the blood specimen to calculate the retraction in­dex which is normally 0.3—0.5.

Capillary permeability. Konchalovsky-Rumpel-Leede sign. A tourni­quet is applied to the forearm and changes occurring in the skin are as­sessed. If petechiae appear on the skin below the tourniquet, the test is positive. Application of a sphygmomanometer cuff and the appearance of more than 1 petechiae on the skin area of 1 cm2 at a pressure of about 100 mm Hg is interpreted in the same way.

Cupping glass test. Air is evacuated from a cup applied to the skin (rarefaction of about 200 mm Hg) for two minutes. If the test is positive, petechiae develop on the skin under the cup. The number of petechiae shows the degree of affection of the vascular wall.

Pinch test. A haemorrhagic spot appears at the site of a pinch, which gradually increases in size and becomes more intense.

Mallet symptom. Ecchymosis develops on the skin after tapping with a percussion mallet.

Determining activity of the 1st phase of blood coagulation. The simplest test is the determination of time of plasma recalcification. The time of coagulation of oxalate plasma, after adding an optimum quantity of calcium chloride to it, is determined. (The oxalate plasma is prepared by mixing 9 parts of plasma with 1 part of a 1.34 per cent sodium oxalate solu­tion and separation of plasma by centrifuging.) The test characterizes blood coagulability in general. Its results somewhat differ from those of the whole blood coagulability tests, in which the formed element factors are also involved. The normal time of recalcification is 60-70 seconds.

The prothrombin consumption test characterizes the activity of those plasma factors which utilize prothrombin in the process of thrombin for­mation. The prothrombin time of plasma (see below) and serum is deter­mined. The higher the prothrombin consumption during plasma coagula-



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Chapter 9. Diseases of the Blood



 


tion, the less is its amount in the serum and the longer it takes to coagulate, and vice versa. It follows that shorter time of prothrombin consumption test indicates disordered formation of thromboplastin.

Determining activity of the 2nd phase of blood coagulation. The activi­ty of the 2nd phase of blood coagulation (formation of thrombin) depends on prothrombin concentration. Its determination is difficult; the overall activity of the prothrombin complex (factors II, V, VI, VII, and X) is therefore established. The method consists in determination of the rate of oxalate plasma coagulation after adding excess thromboplastin and calcium chloride (Quick's time). Since the time of coagulation depends on some conditions (thromboplastin concentration, temperature, etc.), the prothrombin index is usually determined: percentage ratio of the pro­thrombin time of the donor's plasma to the prothrombin time of the pa­tient's plasma (normally it is 80-100 per cent).

Heparin tolerance test characterizes the same phase of coagulation. The test consists in determining the deviation (with respect to norm) in the time of oxalate plasma coagulation after adding heparin with subsequent recalcification. As the activity of the coagulants increases (tendency to thrombosis) the plasma tolerance to heparin increases, and the time of plasma coagulation decreases. If the activity of the anticoagulants predominates (tendency to bleeding), the time increases.

Determining activity of the 3rd phase of blood coagulation. This is the determination of fibrinogen by an equivalent content of fibrin.

Additional tests. Apart from the mentioned relatively simple methods, there are many complicated tests by which the activity of components of the coagulation and anticoagulation systems are determined. Two of them are now commonly used for the determination of the general coagulation tendency of blood (tendency to hypo- or hypercoagulation). The methods are known as thrombotest and thromboelastography.

Thrombotest. A 0.1 ml specimen of oxalate plasma is placed in 5 ml of a 0.5 per cent calcium chloride solution. Sedimentation of fibrin after a 30-minute incubation at 37 °C varies in character (from slight opalescence or the appearance of minutest fibrin grains to the formation of a firm clot), depending on the coagulating properties of blood. Seven degrees of throm­botest are distinguished, from which three correspond to hypocoagulabili-ty, two (4th and 5th) normal coagulability and two (6th and 7th) hyper-coagulability of blood.

Thromboelastography. The test gives a graphic representation of the entire process of spontaneous coagulation of unaltered (native) blood or plasma. A blood specimen is taken from the vein by a silicon-coated needle and placed into a small cell into which a rod bearing a disc is immersed. The cell is vibrated by an electric motor. The disc remains motionless till


the blood specimen remains liquid. As the blood thickens, the disc becomes engaged, and the rod with a mirror attached to it begins vibrating. A beam of light is reflected from the mirror and recorded on a sensitive paper in the form of a zig-zag curve (thromboelastogram). By measuring its separate portions it is possible to assess some properties of the coagulation process, for example, the " reaction time", which corresponds to the length of the 1st and 2nd phases of blood coagulation, the time of clotting (the 3rd phase), elasticity and strength of the clot, and some other indices characterizing hyper- or hypocoagulability of blood (see Appendix).

Summation of the findings of all mentioned tests gives a coagulogram characterizing the condition of the blood coagulation system.

X-RAY EXAMINATION

X-rays can be used to reveal enlargement of the mediastinal lymph nodes (lymphoid leucosis, lymphogranulomatosis, lymphosarcoma) and also changes in the bones which occur in some types of leucosis and malig­nant lymphoma (focal destruction of bone tissue in myeloma, bone destruction in lymphosarcoma, consolidation of bones in osteomyelosclerosis). Changes in the bone tissue are better revealed by X-rays. The spleen is not seen during common X-ray examination. Splenoportography is a special technique which is used for examining the vessels of the spleen (see section on splenoportography).

RADIOISOTOPE METHODS OF STUDY

The spleen function is studied by administering plasma or erythrocytes labelled with radioactive iron (59Fe) into the circulatory system. Foci of erythropoiesis, e.g. in erythraemia and other affections, can be established by this method.

The spleen can also be scanned with the patient's erythrocytes labelled by radioactive chromium (51Cr) or a colloidal solution of gold (198Au) which is captured by the reticuloendothelial cells. This method is suitable for determining the spleen dimensions and for revealing focal affections in it.






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