GEMOGLOBINOMETRIYA (hemoglobin + grech, to measure metreo) — determination of quantity (concentration) of hemoglobin of blood. In a wedge, laboratory practice of G. it is carried out at a research of patients, prof. surveys, examination.
There are three basic groups of methods G.: colorimetric, gas-metric and on the content of iron in blood. Besides, there is a semi-quantitative method of definition of concentration of hemoglobin on the specific weight of blood.
Colorimetric methods the definitions of hemoglobin developed in the basis by H. Welcker, U. Govers, G. Sali in 19 century are based on ability of hemoglobin of blood to give at chemical reactions color derivatives — oxyhemoglobin, carboxyhaemoglobin, a methemoglobin, gemiglobintsianid, muriatic hemin etc.
There are various designs of the devices used at this method G.: from visual, like comparators and colorimeters, to photoelectrometric colorimeters and photometers (see Colorimetry, Photometry, Spektrofotometriya). A number of gemometr (Fleyshlya — Mishera, Byurker, Autenrita, «Migos», «Tseyss-Ikon», Sikka) became outdated. For broad practical work in the USSR release gemometra of GS-Z (a Sahli hemoglobinometer, option No. 3; fig.) representing a small support from dark plastic with three viewing windows. Pipettes are attached to the device, among them there is precisely otgraduirovanny pipette on 20 mkl for blood sampling. In extreme nests of a rack the soldered test tubes with standard solution are located. The graduated test tube with a blood sample, divorced decinormal salt to - that is inserted into an average nest of a support. The graduated test tube has two bench circular marks: lower corresponds to 0,2 ml, and upper — 2 ml. The scale of a test tube is graduated in gram-percent.
Originally the amount of hemoglobin could be designated in so-called units of Sali, blood with contents 5 OOO OOO of erythrocytes in 1 mkl was taken for 80 units. Then these units began to express as a percentage to norm. But authors of different types of gemometr took different concentration of hemoglobin for 100%: from 14 to 17,3 g of %. Therefore sizes of percentage of hemoglobin were incomparable. Transition to expression of concentration of hemoglobin to gram-percent (of %) — amount of hemoglobin in grams in 100 ml of blood is carried out. In the USSR for 100% 16,7 g of % are accepted. Thus, 1 g of % corresponds in percentage terms 6%.
Gemometra of Sali's type demand regular standardization and check as color of standard solutions changes over time. Besides, assessment of results differs in subjectivity, at different researchers discrepancy of indicators can reach 15% and more at the same investigated.
Definition of concentration of hemoglobin gemometry GS-3 (Sali). Necessary reactants: decinormal solution salt to - you and a dist, water. Course of definition: a capillary pipette of a gemometr — Sali's pipette — nasasyvat the blood acting from a puncture of skin to a mark 0,02 (20 mkl) and carefully (not to make foam mix) blow in the graduated test tube of a gemometr with decinormal solution salt to - you, previously poured to the lower circular tag; the capillary is carefully washed out three times in mix, contents of a test tube are mixed a glass rod and the test tube is inserted into an average nest of a gemometr between standards; wait for 5 min. At reaction of hemoglobin with decinormal solution salt to - you are formed muriatic hemin which has brown coloring. Under direct vision add to a test tube a dist, water (a pipette on drops), mixing solution a glass rod and comparing coloring of solution of muriatic hemin in an average test tube to color of the standard in ampoules. When coloring of the examinee of solution matches on color standards, the test tube is removed from a nest of the device, determine by a scale of a test tube a fluid level (on bottom edge of a meniscus of solution) that corresponds to concentration of hemoglobin.
Definition of concentration of hemoglobin a photo-electric eritrogemometr (it is at the same time possible to count quantity of erythrocytes). The method is based on transformation of hemoglobin of blood into oxyhemoglobin. The reactant for times - maintaining blood contains 1 g of sodium carbonate in 1 l of the diart. of water. Course of definition: from 5 ml of a reactant a pipette from a Sahli hemoglobinometer bring in a test tube 40 mkl blood, carefully mix and pour out in a ditch of an eritrogemometr. Measure attenuation range by solution of hemoglobin of waves of a certain length of a blue part of a range according to the management to use of the device. The scale of an eritrogemometr is made in gram-percent.
Definition of concentration of hemoglobin photoelectrocolorimeter of Federal Energy Regulatory Commission-M or Federal Energy Regulatory Commission-56 according to G. V. Derviz and A. I. Vorobyov it is based on transformation of hemoglobin of blood into the oxyhemoglobin which is formed at hemolysis of erythrocytes solution of ammonia. Reactant: 0,024 N (i.e. 0,04%) solution of ammonia. Course of definition: from 4 ml of 0,04% of solution of ammonia Sali's pipette bring in a test tube 20 mkl blood with usual triple rinsing it. The test tube is stirred up and poured out in a ditch. Definition of hemoglobin on Federal Energy Regulatory Commission-M is carried out at the green filter and according to instructions for use the device.
In size of an ekstintion (an indicator of optical density) find the corresponding value of concentration of hemoglobin in gram-percent. The standard curve is formed for each this device on standard solution of hemoglobin.
Definition of concentration of hemoglobin in the colorimeter «Linson Yunior», attached to a tselloskop of AV Ljung Berd (Sweden): hemoglobin from erythrocytes is emitted at hemolysis with solution of a saponin. Reactants: solution for cultivation of blood contains 0,9% solution of sodium chloride — 950 ml, the phosphatic pH M/15 buffer of 7,55 — 45 ml, 35% solution of neutral formalin — 5 ml; 2% solution of a saponin in 0,9% solution of sodium chloride. Solution is stored in the refrigerator no more than two days.
Course of definition: from 4 ml of solution for cultivation of blood bring in a test tube 20 mkl blood, then add 0,1 ml of solution of a saponin, mix, pour out in a ditch of the device and fotometrirut. The table is attached to the device, on a cut the indicator of a scale of optical density is transferred to gram-percent.
Photo-electric gemometra of GF-1, GF-2 and GF-3 with flowing ditches are intended for mass researches in the conditions of laboratory. Scales of these devices are calibrated both for oksigemoglobinovy, and for a gemiglobintsianidny method.
Use of a gemiglobintsianidny method is based on transformation of hemoglobin of blood in gemiglobintsianid at addition to blood of the transforming reactant containing cyanic connections. Reactant: 200 mg of red prussiate of potash, 50 mg of potassium cyanide, 140 mg of monopotassium phosphate and 0,5 ml of Sterox Se (concentrated) dissolve in a dist, to water and bring the volume of solution to 1 l: pH on pH-meter shall be 7,0 — 7,4. The reactant a long time can remain in a large bottle from dark glass, it is desirable in the refrigerator (at t ° 4 °). It is possible to apply also the reactant offered by Drabkin: red prussiate of potash (red blood salt) — 0,2 g, potassium cyanide — 0,05 g, sodium bicarbonate — 1 g, a dist, water — to 1 l.
Course of definition: from 5 ml of a reactant bring in a test tube 20 mkl blood, well mix and leave for formation of a gemiglobintsianid for 2 — 4 min. (during the work with a reactant of Drabkin which has alkali reaction of pH 8,6 and therefore formation of a gemiglobintsianid goes slowly — for 20 min.). Then pour in a ditch and define concentration of hemoglobin on the calibrated photoelectrocolorimeter or the spectrophotometer.
Use of solutions of a tsianmetgemoglobin in a colorimetric method of G. is recognized by the International committee on standardization in hematology by the most exact and objective Method. The specified committee produces reference dolgokhranyashchiysya solution of a gemiglobintsianid for standardization and continuous check and calibration of gemometr.
In the USSR standard (reference) solutions of the tsiansoderzhashchy transforming reactant across Drabkin are produced. Solution of gemiglobin-cyanide is approved as the unified standard for definition of concentration of hemoglobin in clinical diagnostic laboratories.
Gas-metric methods are based on use of property of hemoglobin to attach oxygen or carbon monoxide in strictly certain volumes. Use Van-Slayka's devices, Barkroft (see. Van-Slayka methods ).
Calculation of concentration of hemoglobin for amount of iron in a blood sample. The amount of iron in four humic groups of a molecule of hemoglobin makes 0,347%, therefore, by amount of iron it is possible to establish a hemoglobin content. This group of methods in clinical laboratories is not applied.
The semi-quantitative method (kuprosulfatny) of definition of concentration of hemoglobin on the specific weight of blood was developed by Van-D. D. Van Slyke. Employees of TsOLIPK applied this method to bystry selection of donors. The principle of a method is based that the specific weight of a reactant is chosen according to boundary concentration of hemoglobin, admissible for selection of donors: depending on specific weight the drop of blood emerges or falls by a bottom of a vessel with a reactant that testifies to the reduced or normal concentration of hemoglobin in blood. Preparation of a reactant: on 2 l a dist, waters take 500 g of copper sulfate (pure, for the analysis); the received solution is filtered through a paper fold filter and then, adding small amounts a dist, waters, bring to specific weight 1,052 under control of an urometr, maintaining temperature of solution and water at the level of 20 °. Course of definition: in a glass pour copper sulfate solution 5 cm high. A pure and dry pasterovsky pipette gather blood and from height of 1 cm over the level of solution release a drop in a glass. At first the drop of blood falls by depth of 2 — 3 cm. If in 10 — 15 sec. it emerges or remains weighed in the thickness of solution, then concentration of hemoglobin of blood is lower than 12 g of % if plunges on a bottom of solution — there is higher than 12 g of %. Between the subsequent definitions there have to pass not less than 1 min., during a cut all drops settle on a bottom.
A normal hemoglobin content in blood: for women an average value of 13,0 g of %, fluctuation of 12,0 — 14.0 g of %; for men an average value of 14,5 g of %, fluctuation of 13,0 — 16.0 g of %.
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V. A. Germanov, G. V. Derviz.