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Diagnostics·Clinical hematology tests·Laboratory testing of hemolytic anemia
Summary of the knowledge points in the laboratory testing part of hemolytic anemia in the clinical hematology examination chapter of Diagnostics. Various reasons lead to a type of anemia that shortens the survival time of red blood cells, increases or accelerates their destruction, and the bone marrow hematopoietic function cannot compensate accordingly. .
Edited at 2023-07-09 18:40:36
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Diagnostics·Clinical hematology tests·Laboratory testing of hemolytic anemia
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Diagnostics·Clinical hematology tests·Laboratory testing of hemolytic anemia
concept
definition
A type of anemia that occurs due to various reasons that shorten the survival time of red blood cells, increase or accelerate the destruction of red blood cells, and the hematopoietic function of the bone marrow cannot compensate accordingly.
type
onset and condition
chronic hemolysis
Mostly extravascular hemolysis, onset is slow
Three major characteristics of anemia, jaundice and splenomegaly
acute hemolysis
Sudden onset, massive hemolysis in a short period of time
Causes chills, fever, headache, vomiting, pain in limbs and back, abdominal pain, and hemoglobinuria
In severe cases, acute renal failure, peripheral circulatory failure or shock occurs
Jaundice, paleness, and other signs and symptoms of severe anemia
site of hemolysis
intravascular hemolysis
damage within blood vessels
DIC, mechanical microangiopathy, burns, transfusion, PNH
extravascular hemolysis
Tissue destruction by abundant extravascular monocyte-macrophage system
AIHA, hereditary spherocytosis
in situ hemolysis
globinogenic anemia
Causes and mechanisms
Hemolytic anemia due to intrinsic defects in red blood cells
All intrinsic red blood cell defects are congenital except PNH (acquired)
RBC membrane defects
Hereditary spherocytosis, PNH
RBC enzyme deficiency
G-6PD deficiency
Hemoglobin abnormalities
Abnormalities in the quality of the peptide chain (structural abnormalities)
abnormal hemoglobinopathies
Abnormal amount of peptide chain synthesis (abnormal production)
globinogenic anemia - thalassemia
Hemolytic anemia caused by factors other than red blood cells
Immunity
AIHA, neonatal hemolytic disease, drug-induced red blood cell-related antibodies, blood transfusions with incompatible blood types, etc.
mechanical damage
DIC, TTP, artificial valve, marching hemoglobinuria, etc.
physical factors
Burns, extensive burns, radiation damage, etc.
chemistry medicine
Sulfa drugs, benzene, lead, etc.
biological factors
Malaria, hemolytic streptococcus, mycoplasma pneumonia, leaflets, snake venom, etc.
screening test
Plasma free hemoglobin determination
Reference
<50mg/L(1~5mg/dl)
clinical significance
significantly increased
Intravascular hemolysis occurs (it does not increase when extravascular hemolysis occurs)
Mild increase
Autoimmune hemolytic anemia AIHA, globin production disorder anemia
Serum haptoglobin assay
Reference
0.7~1.5g/L(70~150mg/dl)
clinical significance
reduce
Various hemolysis
significantly reduced
intravascular hemolysis
Extremely reduced (almost undetectable) or absent
Severe intravascular hemolysis (free hemoglobin in plasma >1.3g/L)
decrease or disappear
Liver disease, leaflets, congenital ahaptoglobinemia, etc.
increase height
Infection, trauma, malignant tumors, SLE, GC treatment, oral contraceptives, extrahepatic obstructive jaundice, etc.
Plasma methemoglobin albumin determination
method
biochemical method
Methemoglobin albumin ammonium sulfide → ammonium hemochromogen (wavelength 558nm, with absorption peak) → spectrophotometer detection
Electrophoresis
Cellulose acetate membrane electrophoresis→heme albumin zone
Reference
Negative
clinical significance
Positive – severe intravascular hemolysis
Hemosiderinuria test (Rous test)
principle
Prussian blue reaction
Acidified potassium ferrocyanide solution iron ion → blue ferrous iron
Exfoliated renal tubular epithelial cells contain hemosiderin in urine
Observed under the microscope, dark blue material can be seen in the renal tubular epithelial cells in the urine sediment.
Reference
Negative
clinical significance
Positive for several weeks—chronic intravascular hemolysis
PNH
In the early stage of hemolysis, it may be negative
Red blood cell life span determination (51Cr detection)
principle
Label red blood cells with 51Cr and detect the half-life of red blood cells
The normal half-life of red blood cells is 25~32 days
In hemolytic anemia, half-life <15d
clinical significance
Reliable method to determine hemolytic anemia (half-life <15d)
Detection of red blood cell membrane defects
Red blood cell osmotic fragility test
principle
When red blood cells are exposed to hypotonic sodium chloride solution, the cells gradually swell or even rupture, causing hemolysis.
effect
Determine the resistance of red blood cells to hemolysis by hypotonic sodium chloride at different concentrations [osmotic fragility of red blood cells], expressed as the minimum resistance and maximum resistance of the tested red blood cells.
Reference
Hemolysis begins
0.42~0.46% (4.2~4.6g/L) NaCl solution
Complete hemolysis
0. 28~0.34% (2.8~3.4g/L) NaCl solution
clinical significance
Increased brittleness
Hereditary spherocytosis (common)
Warm-body autoimmune hemolytic anemia, hereditary elliptocytosis
Standard - when hemolysis begins and when hemolysis is complete, the sodium chloride concentration is at least two tubes (≥0.04%) ahead of the normal control.
Begins hemolysis>0.5%
Complete hemolysis>0.38%
reduced brittleness
Thalassemia (common)
Iron deficiency anemia, some forms of cirrhosis, obstructive jaundice
Red blood cell incubation osmotic fragility test
principle
During the incubation of red blood cells, glucose is consumed ↑ and the ATP reserve is ↓
ATP↓→The active transmission of cations by the red blood cell membrane is blocked→Nadium accumulates in the red blood cells→The cells swell and the osmotic fragility increases
Reference
Not incubated
50% hemolysis, 4~4.45g/L NaCl
37℃, incubate for 24h
50% hemolysis, 4.65~5.9g/L NaCl
clinical significance
For the diagnosis and differential diagnosis of mild hereditary spherocytosis and hereditary non-spherocytic hemolytic anemia
increased brittleness
Hereditary spherocytosis, hereditary elliptocytosis, hereditary non-spherocytic hemolytic anemia
reduced brittleness
Globin production disorder anemia, iron deficiency anemia, leucocytic anemia, post-splenectomy
Autohemolysis test and correction test
principle
Patients with congenital non-spherocytic hemolytic anemia → Enzyme deficiency in red blood cells and glucose glycolysis disorder → Inability to provide sufficient ATP and maintain the sodium pump function in red blood cells
The patient's red blood cells were incubated in their own plasma for 48 hours under sterile conditions → ATP reserve ↓, the sodium pump function weakened → hemolysis increased
During the incubation process, add glucose and ATP as corrective substances, and use sodium chloride solution as a control to observe whether the hemolysis is corrected.
Reference
Normal people, incubate for 48 hours
Slight hemolysis, hemolysis degree <3.5%
For normal people, add glucose and ATP and incubate for 48 hours.
Hemolysis was significantly corrected, and the hemolysis degree was <1%.
clinical significance
Differential diagnosis of hereditary spherocytosis and congenital non-spherocytic hemolytic anemia
hereditary spherocytosis
Hemolysis was significantly enhanced after incubation
After adding glucose and ATP, hemolysis was significantly corrected.
Congenital non-spheroid hemolytic anemia type I (G-6-PD defect)
After incubation, autohemolysis worsens
After adding glucose and ATP, the hemolysis was partially corrected.
Congenital non-spheroid hemolytic anemia type I (pyruvate kinase PK deficiency)
After incubation, autohemolysis was significantly enhanced
Add glucose and incubate, hemolysis cannot be corrected
Add ATP and incubate to correct hemolysis
Detection of red blood cell enzyme deficiencies
concept
Hemolytic anemia/erythroenzymopathy caused by red blood cell enzyme deficiency
A group of diseases in which hemolysis occurs due to genetic defects in enzymes involved in red blood cell metabolism (mainly sugar metabolism), resulting in changes in activity.
Methemoglobin reduction test
principle
Sodium nitrite is added to the blood being tested → Hb is converted into high iron Hb (brown)
High iron Hb→NADPH methemoglobin reductase→ferrous Hb
When the G-6-PD content and activity are normal, the amount of NADPH is sufficient to complete the above reaction (when it is insufficient, the reduction speed slows down or cannot be reduced)
Reference
Methemoglobin reduction rate>75%
Methemoglobin 0.3~1.3g/L
clinical significance
reduce
Favismosis, primaquine-type drug-induced hemolytic anemia
Cyanide-ascorbic acid test
principle
Sodium ascorbate HbO2→H2O2
Sodium cyanide → inhibits H2O2 enzyme and inhibits the decomposition of H2O2
H2O2 GSH→GSSG
GSSG NADPH→GSH
G-6-PD defect→NADPH↓→GSH↓→H2O2 accumulation→ferrous Hb is reduced to high-iron Hb
clinical significance
normal human blood
≥4h, turn brown
Homozygous G-6-PD deficiency
≤2h, turns brown
Heterozygous G-6-PD deficiency
3~4h, turns brown
Denatured globin body production test
principle
G-6-PD deficiency→GSSG↓, high iron Hb↑→denatured globin bodies in red blood cells
G-6-PD deficient blood and control specimens: acetyl phenylhydrazine, incubate at 37°C for 2~4 hours → thin blood slices, stained with 1% brilliant tar blue
Calculate the percentage of red blood cells containing 5 or more globin bodies
Reference
<30%
clinical significance
>45%
G-6-PD deficiency, unstable Hb, HbH disease and other denatured globin bodies
G-6-PD fluorescence spot test and activity assay
principle
G-6-PD NADP→NADPH
NADPH, can fluoresce under ultraviolet light (absorption peak, at wavelength 340nm)
G-6-PD activity is measured by the amount of NADPH produced per unit time.
Reference
Normal people have very strong fluorescence, and the enzyme activity is (4.97±1.43) U/gHb
clinical significance
Very weak or no fluorescence
G-6-PD defects
Light to moderate fluorescence
Heterozygotes, certain G-6-PD variants
PK Fluorescence Screening Assays and Activity Assays
principle
In the presence of ADP, PK can catalyze enol phosphate pyruvate into pyruvate
In the presence of NADH, pyruvate LDH → lactic acid
When lactic acid is formed, the autofluorescent NADH→NAD disappears.
Reference
PK activity is normal, fluorescence disappearance time ≤20min
Enzyme activity (15.1±4.99) U/gHb
clinical significance
Fluorescence disappearance time>60min
Severe PK deficiency (homozygous)
Fluorescence disappearance time 25~60min
PK deficiency (heterozygous)
Detection of abnormal globin production
Hemoglobin electrophoresis
principle
The basic principle is the same as serum protein electrophoresis
Reference
normal person
The electrophoresis pattern shows 4 zones, from anode to cathode: HbA (large amount) → HbA2 (low amount) → non-hemoglobin components NH1 and NH2 (less amount)
clinical significance
Increased HbA2
Important basis for diagnosing beta thalassemia minor
Individual pernicious anemia, megaloblastic anemia caused by folic acid deficiency, some unstable hemoglobinopathies
HbA2 reduction
Iron deficiency anemia, sideroblastic anemia
Fetal Hb acid elution test
principle
HbF acid resistance>HbA
The fixed blood smear is moistened in an acidic buffer for a certain period of time so that only red blood cells containing HbF are not eluted, and then stained with eosin to turn bright red.
clinical significance
Physiological
Positive
Cord blood, newborn, infant
<1%
aldult
pathological
A few red blood cells are positive
Thalassemia patients with mild disease (heterozygotes)
A large number of red blood cells are positive
Severe thalassemia patients
Fetal Hb determination or HbF alkaline denaturation test
principle
In alkaline solution, HbF is not easily denatured and precipitated, while other Hb is easily denatured and precipitated. Determine the Hb content in the filtrate.
Reference
Adult<2%
Newborns 55~85%
About 1 year old ≈ adult
clinical significance
significantly increased
beta thalassemia
Light height increase
Acute leukemia, aplastic anemia, pure erythroleukemia, lymphoma
HbA2 quantitative determination
Reference
1~3.2%
clinical significance
Same as hemoglobin electrophoresis
Autoimmune hemolytic anemia testing
antiglobulin test
principle
Antiglobulin test positive
Incomplete antibodies that have bound to the corresponding antigen on red blood cells are unable to connect, or bridge 2 adjacent red blood cells without exhibiting red blood cell agglutination.
Antiglobulin antibodies can bind to the Fc segments of multiple incomplete antibodies and act as a link or bridge, causing red blood cell aggregation to be observed.
effect
Positive direct Coombs test - incomplete antibodies have been bound to the surface of red blood cells
Positive indirect Coombs test - incomplete antibodies are present in the patient's serum
Reference
Both direct and indirect antiglobulins were negative.
clinical significance
Positive
Hemolytic disease of the newborn, autoimmune hemolytic anemia, SLE, rheumatoid arthritis, certain lymphomas, methyldopa and penicillin-type and other drug-induced hemolytic reactions
Warm antibodies, cold antibodies
AIHA (mostly warm antibody type, IgG)
AIHA (a small number of them belong to the cold antibody type, IgM)
If necessary, conduct the test at 4°C to eliminate false negative reactions.
Antibody subtype
AIHA, mostly IgG type, a few are IgG C3 type, C3 type, IgA, IgM type, very few IgG subtypes, etc.
Broad-spectrum antiglobulin serum should be used for testing, and monovalent antisera of various subtypes should be added if necessary to increase the positive rate of detection.
Indirect Coombs test
For the detection of Rh or ABO pregnancy immune hemolytic disease of the newborn and incomplete antibodies in maternal serum
Cold agglutinin test
principle
Cold agglutinin is a reversible antibody
At low temperatures, agglutination can occur with own red blood cells, "O" type red blood cells, and red blood cells of the same type as the patient's.
When the temperature increases, the agglutination phenomenon disappears
Reference
The titer is <1:40, and the optimal reaction temperature is 4℃
clinical significance
Cold agglutinin has high potency
Certain AIHA
Hot and cold hemolysis test
principle
There is a special hemolysin in the serum of PCH patients
At 0~4°C, hemolysin combines with red blood cells and adsorbs complement, but does not dissolve hemolysis.
But when the temperature rises to 30~37℃, hemolysis occurs
Reference
Negative
clinical significance
Positive
Paroxysmal cold hemoglobinuria PCH
Certain viral infections (measles, mumps, chickenpox, flyers, etc.)
Paroxysmal nocturnal hemoglobinuria PNH related testing
concept
nature
Chronic intravascular hemolysis caused by acquired red blood cell membrane defects
Performance
Aggravated during sleep, accompanied by episodic hemoglobinuria and pancytopenia
Sucrose hemolysis test
principle
The ion concentration of sucrose solution is low, and incubation can strengthen the binding of complement to the red blood cell membrane → small holes can be formed on the red blood cell membrane of PNH patients → sucrose enters the red blood cells → the osmotic pressure within the red blood cells changes → hemolysis
Reference
Negative
clinical significance
mildly positive
Partial megaloblastic anemia, aplastic anemia, AIHA, hereditary spherocytosis
effect
Screening test for PNH
Acidification hemolysis test (Ham test)
principle
The red blood cells of PNH patients have increased sensitivity to complement → incubate in acidified serum (pH 6.6~6.8) at 37°C → hemolysis
Features
Sensitive, fewer false positives
Reference
Negative
clinical significance
Diagnosis of PNH (for secondary diagnosis in patients with positive sucrose hemolysis test)
Severe attack of AIHA (rare cases)
Snake venom factor hemolysis test
principle
A protein with M=144000 extracted from cobra venom → directly activates C3 in serum → activates complement through the alternative pathway → hemolysis occurs in PNH patients
clinical significance
Specific PNH test