151
`
`Definition
`
`Hemoglobin (Hb) is the protein contained in red blood cells
`that is responsible for delivery of oxygen to the tissues . To
`ensure adequate tissue oxygenation, a sufficient hemoglo-
`bin level must be maintained . The amount of hemoglobin
`in whole blood is expressed in grams per deciliter (g/dl) .
`The normal Hb level for males is 14 to 18 g/dl ; that for
`females is 12 to 16 g/dl . When the hemoglobin level is low,
`the patient has anemia . An erythrocytosis is the consequence
`of too many red cells ; this results in hemoglobin levels above
`normal .
`The hematocrit measures the volume of red blood cells
`compared to the total blood volume (red blood cells and
`plasma) . The normal hematocrit for men is 40 to 54% ; for
`women it is 36 to 48% . This value can be determined directly
`by microhematocrit centrifugation or calculated indirectly .
`Automated cell counters calculate the hematocrit by mul-
`tiplying the red cell number (in millions/mm3) by the mean
`cell volume (MCV, in femtoliters) . When so assayed, it is
`subject to the vagaries inherent in obtaining an accurate
`measurement of the MCV (see Chapter 152) .
`Both the hemoglobin and the hematocrit are based on
`whole blood and are therefore dependent on plasma vol-
`ume . If a patient is severely dehydrated, the hemoglobin
`and hematocrit will appear higher than if the patient were
`normovolemic ; if the patient is fluid overloaded, they will
`be lower than their actual level . To assess true red cell mass,
`independent radionuclide evaluation of the red cells and
`plasma (by 51 Cr and 131I respectively) must be performed
`.
`
`Technique
`
`Hematocrit
`
`If the hematocrit must be determined quickly, as is often
`the case when a patient hemorrhages, it may be necessary
`to measure the hematocrit directly without the use of an
`automated counter. The materials needed are :
`
`Lancets
`•
`•
`Alcohol prep pads
`Gauze pads
`•
`• Microhematocrit tubes (heparinized)
`• Sealant ("Seal-Ease," "Crit-Seal," etc)
`Microhematocrit centrifuge
`•
`Microhematocrit reader
`•
`If venipuncture is required : tourniquet, syringe, tube
`•
`containing anticoagulant (EDTA, citrate)
`
`For hematocrits obtained byfingerstick, wipe the fingertip
`pad of the fourth finger of the nondominant hand with the
`alcohol prep pad . Make certain the area is allowed to dry .
`Prick the fingertip with the lancet . Place the hematocrit tube
`
`LABORATORY
`
`Hemoglobin and Hematocrit
`
`HENNY H. BILLETT
`
`near the incision site and allow the blood to flow via capillary
`action into the hematocrit tube until it is two-thirds to three-
`fourths full or to a predesignated mark on the tube . Avoid
`"milking" the finger if possible ; this causes the expression
`of tissue fluids and may result in a falsely low hematocrit.
`Always fill at least three tubes . For hematocrits obtained by
`venipuncture, draw a sample of blood into the tube contain-
`ing anticoagulant and mix well . Dip the hematocrit tube
`into the blood and allow the blood to rise to the desired
`two-thirds to three-quarters level . Because blood cells nat-
`urally sediment, a prior thorough mixing of the blood in
`the tube is necessary to ensure accurate reading .
`After cleaning the outside of the hematocrit tubes of
`excess blood, invert the tube slowly so that the blood mi-
`grates just short of the bottom end of the tube . Seal the
`bottom of the tube with sealant . Make certain that little or
`no air is interspersed in the column of blood . If the seal is
`incomplete, leakage will occur during centrifugation and
`false readings will be obtained .
`Place the tubes in a microhematocrit centrifuge and spin
`for 3 to 5 minutes at high speed . A shorter spin will not
`allow for complete sedimentation .
`Using either a hematocrit reader or any ruled apparatus,
`measure the length of the column of the packed red cells
`and divide it by the length of the whole column of blood
`(cells and plasma), as in Figure 151 .1 . To obtain the he-
`matocrit, multiply this number by 100% . Average all read-
`ings obtained from the different microhematocrit tubes .
`Example : If the column of packed red cells measures 20
`mm and the whole blood column measures 50 mm, the
`hematocrit is 20/50 = 0 .4 or (0 .4 x 100%) = 40% .
`
`Hemoglobin
`
`Hemoglobin determinations will usually be performed by
`an automated cell counter from a tube of well-mixed EDTA-
`anticoagulated blood filled to a predetermined level . In this
`assay, all forms of hemoglobins are converted to the colored
`
`Figure 151 .1
`Microhematocrit tube after sedimentation . The hematocrit is a ratio
`of the packed cells to total volume .
`
`7 1 8
`
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`1 5 1 . HEMOGLOBIN AND HEMATOCRIT
`
`719
`
`protein cyanomethemoglobin and measured by a colorim-
`eter. An inadequate sample, whether due to insufficient
`volume or inadequate anticoagulation, may give false read-
`ings . If it is necessary to determine the level of anemia
`quickly, the hematocrit is an easier, more convenient test .
`
`Hemoglobin Electrophoresis
`
`Hemoglobin electrophoresis measures the mobility of
`hemoglobin in an electric field ; it can therefore detect only
`those abnormalities in hemoglobin that alter the charge .
`Electrophoretic mobilities are affected by pH and by the
`medium in which the test is conducted . Screening tests typ-
`ically use a hemolysate of anticoagulated blood electrop-
`horesed on cellulose acetate at pH 8 .6 to 8 .8 . If necessary,
`a further electrophoresis in starch gel at pH 6 .2 to 6 .8 is
`performed . At that stage, the work will usually be per-
`formed by a specialized laboratory .
`Hemoglobin electrophoresis will not readily assess situ-
`ations where there are neutral amino acid substitutions or
`where the hemoglobin is normal but the constituent chains
`are not produced in equal numbers (thalassemias) . The di-
`agnosis of alpha thalassemia of a mild to moderate degree
`cannot be made by hemoglobin electrophoresis ; the diag-
`nosis of beta thalassemia may be made by inference from
`an increase in the Hb A 2 .
`A standard electrophoresis would look like Figure 151 .2 .
`
`Basic Science
`
`The molecular weight of hemoglobin is approximately 64,500
`daltons . Hb is composed of two pairs of dissimilar chains,
`a and (3, each defined by a specific amino acid sequence
`and incorporating an iron-containing heme group . Two a-
`(3 dimers combine to form a hemoglobin tetramer . This
`allows for the "heme-heme" interaction necessary for ef-
`fective oxygen uptake (deoxyhemoglobin -> oxyhemoglo-
`bin) and delivery (oxyhemoglobin -* deoxyhemoglobin) .
`The oxygen affinity of hemoglobin is a function of this
`heme-heme interaction and of pH (Bohr effect), and is a
`measure of how many hemoglobin molecules have oxygen
`bound to them for a given level of oxygen tension . In a
`normal individual the major hemoglobin is Hb A, consti-
`tuting approximately 97% of the total hemoglobin . Varia-
`tions and/or amino acid substitutions in these chains exist .
`Some are deleterious to the normal function of hemoglobin,
`whereas others may have relatively normal oxygen affinity
`and stability . Hemoglobins containing different types of
`chains make up the remainder of the hemoglobin content
`in red cells (a2b2 = Hb A2 approximately 2% ; a2 y 2 = Hb F
`approximately 1%) .
`Substitutions in the normal hemoglobin amino acid se-
`quence may result in hemoglobins that have different sub-
`unit interactions and varying affinities for oxygen . For
`example, a substitution of the sixth amino acid on the beta
`chain causes Hb S, or sickle hemoglobin . Hb S has a lower
`oxygen affinity and surrenders its oxygen more readily . Hb
`F, a normal minor hemoglobin constituent, has a higher
`oxygen affinity.
`
`Figure 151 .2
`A standard hemoglobin electrophoresis (cellulose acetate, pH 8 .6) .
`
`If the oxygen dissociation curve is abnormal, the body
`will adjust the hemoglobin level to ensure adequate oxygen
`distribution to the tissues . Thus in a rare disease like hemo-
`globin Hotel Dieu, the difficulty in extracting oxygen from
`a variant hemoglobin with increased oxygen affinity could
`result in a lack of oxygen for the tissues (tissue hypoxia)
`and a compensatory erythrocytosis . The smaller fraction of
`oxygen released from the hemoglobin is thereby offset by
`the increased number of hemoglobin molecules . Similarly,
`in sickle cell anemia, the decreased oxygen affinity allows
`these patients more tissue oxygen at any given hemoglobin
`level .
`
`Clinical Significance
`
`Many anemias are detected by routine laboratory screening
`performed before the patient is symptomatic . When the
`patient does have symptoms from an abnormality in the
`hemoglobin level, the symptoms are often a nonspecific
`weakness or fatigue . The only finding on physical exami-
`nation may be pallor ; additional changes in the nail beds
`(such as spooning), glossitis (red tongue), or hepatosple-
`nomegaly (enlarged liver or spleen) may give a clue to the
`etiology of the anemia . Symptoms are usually related to the
`level of hemoglobin, its abruptness of onset and its duration .
`A patient with pernicious anemia may feel well at the same
`level of hemoglobin that would cause severe weakness in a
`patient with acute gastrointestinal hemorrhage . This is due
`to volume compensation by plasma and shifts in the oxygen
`dissociation curve which occur over time .
`When first confronted with an abnormal hemoglobin or
`hematocrit level, the next step is to assess the red cell indices
`(see Chapter 152), peripheral smear (Chapter 155), and the
`reticulocyte count (Chapter 156) in light of the patient's
`history and physical examination .
`
`References
`
`Adamson] W, Finch CA. Hemoglobin function, oxygen affinity and
`erythropoietin. Annu Rev Physiol I975 ;37 :351 .
`Bunn HE . Hemoglobin I . Structure and function . In : Beck WS,
`Hematology. Cambridge, MA : MIT Press, 1981 ;129.
`Scott AF et al. The molecular basis of hemoglobin. Am J Hum
`Genet I98I ;33 :I29 .
`Wallerstein RO. Laboratory evaluation of anemia . West J Med
`1987 ;I46 :443 .
`
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`

`Clinical Methods
`
`THIRD EDITION
`The History, Physical, and Laboratory
`Examinations
`
`Edited by
`H. KENNETH WALKER, M.D.
`Professor of Medicine and Associate Professor of Neurology
`Emory University School of Medicine
`Atlanta, Georgia
`W. DALLAS HALL, M.D.
`Professor of Medicine
`Emory University School of Medicine
`Atlanta, Georgia
`J. WILLIS HURST, M.D.
`Candler Professor of Medicine (Cardiology)
`Emory University School of Medicine
`Atlanta, Georgia
`
`With 193 contributing authors
`
`Illustrations by Leon Schlossberg and Charles H. Boy ter
`
`Butterworths
`Boston London Singapore Sydney Toronto Wellington
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`The History,
`Physical, and
`Laboratory
`Examinations
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`H. Kenneth Walker
`W. Dallas Hall
`J. WaUs Hurst
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