LABORATORY FINDINGS

Findings of Haemolysis

• Increased unconjugated (indirect) Bilirubin (2.5 mg/dl - 5.0 mg/dl), decreased haptoglobin, slight increase in plasma haemoglobin.
• Occasionally massive haemoglobinemia, haemoglobinuria, and haemosiderinuria in fulminant disease and in paroxysmal cold haemoglobinuria.

• Moderately severe anaemia, increased reticulocytosis unless there is impaired marrow function, anisocytosis, nucleated RBC; marked polychromatophilia and increased MCV reflect increased numbers of reticuloyctes
• Warm type - microspherocytes due to piecemeal ingestion of antibody-coated RBC membrane by macrophages in the RE system.  Minimal autoagglutination, which is not enhanced by cooling.
• Cold type - clumping of RBC on cooling and even at room temperature; clumps dissolve upon warming to 37 degrees C.
• Leukocytosis - may be slight but might reach leukemoid proportions (<50,000/ul) as a result of stress response to acute haemolysis

• Marked hypercellularity - result of normoblastic hyperplasia in response to haemolysis
• Increased iron deposits - result of accelerated RBC turnover
• Megaloblasts may occur;  these are secondary to folic acid deficiency following prolonged severe haemolysis
• Underlying lymphoma or lymphocytic leukaemia may be present.

A positive direct Coombs' test indicates that an antibody is attached to the surface of the patient's RBC.  This will be indicated by broad-spectrum antiglobulin reagents.  Using monospecific Coombs' reagents, warm AIHA will react maximally at 37 degrees C with anti-human IgG serum along (30% - 40% of patients) or with both anti-IgG and anticompolement sera (40% - 50% of patients).  When only IgG is detected, it is usually an antibody reacting with Rh antigenic sites.  These sites are separated so that two IgG molecules will not be close enough to activate the complement system.  When IgG and complement are both detected, the antibody is usually directed against non-Rh antigens that are spaced so that two IgG molecules can fix complement.

Cold AIHA reacts maximally at 4 degrees C and reacts only with anti-complement serum.  The IgM antibody is rarely detected it quickly separates from RBC, especially on warming.  The bound complement may be detected at 37 degrees C.

Indirect Antiglobulin (Coombs1) Test

The indirect Coombs' test indicates the presence of free autoantibody in the patient's serum, which occurs when there are large amounts of autoantibody and low RBC binding affinity.  Warm autoantibodies are usually IgG, they agglutinate test cells at 37 degrees C, and they show no increase in reactivity at 4 degrees C.  Cold autoantibodies are IgM, they strongly agglutinate test cells at 4 degrees C, but they may sometimes react at up to 33 degrees C.  This indicates a wide thermal range of reactivity.

Eluates of RBC

In warm AIHA, antibody eluted from the RBC surface is usually the same as that identified in the patient's serum in the indirect Coombs' test.  In cold AIHA, no antibody can be eluted from RBC surface because the antibody usually has already eluted spontaneously.

Cold Agglutinin Titre

See Table 8-1 for examples of conditions showing increased cold agglutinin titers.

Other Laboratory findings
 

• Positive Donath-Landsteiner test in paroxysmal cold haemoglobinuria.  An IgG antibody initially adsorbs to the surface of RBC along with complement at a low temperature.  The antibody then causes intravascular haemolysis and haemoglobinuria as the temperature is increased to 37^odegrees C.  This antibody is termed a biphasic haemolysin.

• Decreased serum complement - this is usually associated with cold AIHA because IgM, which occurs in this disorder, binds complement.  Decreased complement complement also occurs in paroxysmal cold haemoglobinuria during an attack because the Donath-Landsteiner antibody binds complement.  complement is occasionally decreased in warm AIHA.

• Positive antinuclear antibody suggests lupus erythematosus as the basis of warm AIHA.
• Increased osmotic fragility test - this reflects the presence of spherocytes which usually occur in warm AIHA;  the osmotic fragility is not enhanced by incubation at 37 degrees C.
• Increased autohaemolysis test - this occurs during periods of active haemolysis;  it is not corrected by the addition of glucose.

LABORATORY FINDINGS

• Normochromic anaemia - severity is variable
• Macrocytosis (MCV 105 um3 - 110 um3) - reflects reticulocytosis
• Findings of haemolysis (increased unconjugated bilirubin, reticulocytosis, increased LDH; decreased haptoglobin) - usually brief, stopping when the drug is withdrawn
• Positive direct Cooms' test - this finding is essential to establish an immune basis of the haemolysis.  The adsorbed globulin may be IgG or complement,  depending on the mechanism:10
• Immune complex - usually complement only
• Drug adsorption - IgG or IgG and complement
• Warm autoimmune haemolytic anaemia - IgG only;  strongly positive
• Nonimmunologic protein adsorption - IgG and complement (and other serum proteins)
Antibody identification:
• Immune complex - serum usually reacts with RBC in presence of drug; eluate nonreactive
• Drug adsorption - serum reacts with drug-treated RBC to high titre; eluate reacts with drug-treated RBC
• Warm autoimmune haemolytic anaemia - antibody in serum and eluate is similar to that found in warm antibody AIHA
• Nonimmunologic protein adsorption - no serum antibody; eluate nonreactive

LABORATORY FINDINGS

Peripheral Blood

• This characteristically shows burr and fragmented RBC and occasional nucleated RBC
• Decreased Hb - often < 6 g/dl
• Increased reticulocytes - reflects bone-marrow response to haemolysis

• Absent serum haptoglobin - reflects chronic haemolysis
• Findings of disseminated intravascular coagulation - increased fibrin split products and decreased platelet count
• Urine moderate to massive protein, RBC, WBC; granular, hyaline, and RBC casts
• Azotemia - BUN frequently > 100 mg/dl
• Oliguria

LABORATORY FINDINGS

Peripheral Blood

• Normocytic, normochromic anaemia, moderate to severe - Hb often < 6 g/dl
• Increased macrocytes and polychromatophilia - reflect reticulocytosis
• Microcytic, hypochromic anaemia - iron deficiency results from recurrent haemoglobinuria
• Decrease in platelets (approximately 67%) - reflects marrow hypoplasia
• Decreased WBC (approximately 60% of patients) - reflects marrow hypoplasia

• Normoblastic hyperplasia
• Lack of iron late in course of the disease
• Possible aplasia or leukaemia

• Findings of haemolysis - reticulocytosis (10% - 35%); decreased haptoglobin;  increased unconjugated (indirect) bilirubin and LDH; increased urine haemosiderin and Hb; haemoglobinemia, especially during sleep
• Decreased leukocyte alkaline phosphatase reflects dysplastic WBC.
• Decreased serum iron - result of continuous urinary loss of iron
• Abnormal autohaemolysis test, which probably reflects lysis due to complement
• Negative direct Cooms' test i ndicates the nonimmunologic basis of the haematologic disorder.

• These demonstrate lysis of complement-senstive RBC.
• Positive sucrose haemolysis test - in this screening test a sucrose solution of low ionic strength maintains osmotic equilibrium while complement fixes to and haemolyses paroxysmal nocturnal haemoglobinuria erythrocytes.  This test is sensitive but non specific.
• Positive acid haemolysis test (Ham test) (definitive test) - paroxysmal nocturnal haemoglobinuria erythrocytes haemolyse when suspended in fresh, normal, compatible complement-containing serum acidified to pH 6.8.  Verified pH is critical for optimal complement activation.  Spherocytes will also haemolyse and will give a false-positive test.

LABORATORY FINDINGS

Rh Incompatibility

• Foetal blood group Rho(D) - positive
• Maternal blood group Rho(D) - negative
• Positive direct antiglobulin test on cord RBC indicates maternal antibody which is attached to foetal RBC.  Antibody should be eluted and identified.
• Maternal antibody screening positive - the antibody must match that eluted from foetal RBC.  The antibody titre does not correlate with the severity of HDN.
• Normal cord Hb is 14 g/dl - 20 g/dl.  Capillary blood may be up to 4 g/dl higher.
• Cord blood Hb indicates the degree of anaemia and reflects the severity of the disease.
• Macrocytic anaemia (8 g/dl - 16 g/dl) - not usually present at birth, maximal at 3-4 days
• Peripheral blood:  increased nucleated RBC (>10/100 WBC) - this reflects the rapid formation and release from the marrow, liver, and spleen of immature RBC in response to the RBC destruction.  Macrocytes and polychromatophilia reflect reticulocytosis.
• Reticulocytosis (10% to 60%) reflects marrow response to haemolysis.
• Leukocytosis (15,000/ul - 30,000/ul) reflects marrow response to stress.
• Increased serum unconjugated (indirect) bilirubin - present at birth or very shortly thereafter; rises rapidly

• Foetal blood group A or B
• Maternal blood group O
• Weak direct antiglobulin test on cord RBC, which becomes negative within 12 hours after birth.  the weak reaction is due to the small number of antibody molecules attached to the RBC.
• Eluted IgG, anti-A, or anti-B from infant's RBC are tested against adult A and B RBC.
• Anti-A may be found in Group A infant's serum; anti-B may be found in Group B infant's serum.
• Marked microspherocytosis - not seen in Rh haemolytic disease
• Increased osmotic fragility reflects presence of spherocytes
• Increased unconjugated (indirect) bilirubin - this is less elevated and of shorter duration than in Rh incompatibility
• Minimal anaemia

LABORATORY FINDINGS

Peripheral Blood

• Normocytic, normochromic anaemia (Hb < 9g/dl); occasional macrocytosis
• Leukopenia (<2,000/ul) - granulocytes especially decreased; 70% - 90% lymphocytes
• Thrombocytopenia (<70,000/ul) - decrease persists long afgter WBC and RBC return to normal levels
• Decreased reticulocytes

• Aspiration and biopsy - several biopsies may be required because there may be foci of normal cellularity
• Decreased cellularity, especially granulocytes and megakaryocytes
• Increased fat is a chracteristic finding.
• Focal hypercellularity showing relative lymphocytosis (60% - 100%).
• Usually increased iron in RE cells.

• Increased serum iron due to impaired marrow utilisation of iron
• Decreased serum iron-binding capacity
• Increased transferrin saturation, marked (may be almost 100%) - this is often the first sign of reduced marrow utilisation of iron
• Increased serum and urine erythropoietin - levels are  higher than in most other types of anaemia
• Prlonged bleeding time and poor clot retraction reflect thrombocytopenia.
• Increased infections reflect impaired defences associated with severe granulocytopenia.

LABORATORY FINDINGS

Peripheral Blood

• Normocytic to slightly macrocytic anaemia (Hb 7 g/dl - 10 g/dl)
• Increased RBC distribution width indicates two RBC populations; most RBC are normochromic, and some are hypochromic.
• Marked anisocytosis with many bizarre forms
• Occasional basophilic stippling, siderocytes, target cells, normoblasts
• Usually normal reticulocytes - this is inappropriately low for the degree of anaemia
• Normal WBC and platelet counts;  occasionally decreased WBC, especially neutrophils
• Decreased WBC alkaline phosphatase (50% of patients)

• Increase in ringed sideroblasts (<25% of nucleated RBC) - characteristic finding
• Normoblastic hyperplasia, marked, with a shift to younger forms;  occasional megaloblastoid maturation
• Increased stainable iron (haemosiderin)

• Increased serum iron and normal or decreased total iron-binding capacity
• Increased transferrin saturation
• Increased serum ferritin

Non-heme iron accumulates in RBC mitochondria surrounding the nucleus instead of cytoplasmic ferritin. A ring of iron is seen as opposed to the normal situation where only a few (less than 4) small aggregates of iron (ferritin) are present

LABORATORY FINDINGS

Peripheral Blood

• Normochromic, normocytic anaemia (Hb > 9 g/dl)
• Nucleated RBC, even in absence of anaemia;  this suggests marrow invasion
• Marked anisocytosis and poikilocytosis;  "teardrop" erythrocytes occur with myelofibrosis;  polychromatophilia, basophilic stippling
• WBC are normal to decreased;  occasional immature cells suggest marrow invasion
• Platelets are normal to decreased; giant platelets or fragments of megakarocytes suggest marrow invasion.

marrow blood smear showing infiltration by Hodgkin's lymphoma. The large binucleated lymphoma cell is the Reed-Sternberg cell.

• Bone marrow shows the infiltrates of leukaemia, metastatic cancer, fibrosis or plasma cells.  Marrow biopsy is usually more diagnostic than is a marrow aspirate.

• See diseases discussed above.

LABORATORY FINDINGS

• Prolonged activated partial thromboplastin time (APTT) when factor VIII is below 25% - 30%; corrected by normal plasma
• Normal prothrombin time, bleeding time, thrombin time, fibrinogen
• Decreased AHF - definitive test
• Normal von willebrand antigen and ristocetin-induced platelet agglutination
• Possible presence of antibody against AHF - reflects reaction to repeated administration of AHF

Normal / Von Willebrands

LABORATORY FINDINGS Decreased AHF (6% - 60% of normal levels) - occurs in less than 50% of patients Marked increase in AHF following transfusion of normal plasma. Decreased von Willebrand's factor (factor VIII related antigen) - characteristic finding Prolonged bleeding time reflects level of von Willebrand's factor. Decreased platelet retention by glass bead columns Decreased or absent ristocetin - induced platelet agglutination - characteristic finding.  In the presence of normal plasma, the patient's platelets aggregate with ristocetin;  the patient's plasma impairs agglutination of normal platelets in the presence of ristocetin. Normal platelet agglutination with adenosine diphosphate (ADP), epinephrine, and collagen Abnormal activated partial thromboplastin time if AHF is less than 25% - 30% of normal level Normal prothrombin time, fibrinogen, thrombin time, platelet count, and clot retraction

LABORATORY FINDINGS

• Decreased factor IX level - titre <5% in severe cases;  titre > 30% in mild cases
• Prolonged APTT - corrected by aged plasma, normal plasma, and normal serum
• Normal prothrombin time, bleeding time, thrombin time, fibrinogen

LABORATORY FINDINGS

• Increased fibrin or fibrinogen degradation products - these reflect lysis and breakdown of fibrin or fibrinogen;  this finding is necessary for definitive diagnosis
• Decreased platelets
• Decreased fibrinogen - may be normal if the prior level was elevated
• Prolonged thrombin time - only if fibrinogen < 75 mg/dl

• Prolonged prothrombin time and APTT
• Schistocytes on peripheral blood smear
• Findings of intravascular haemolysis - haemoglobinemia, increased LDH, decreased haptoglobin

DIC - Click on image for link to source
	Click on Images for links to case study and image souce The patient is a 20-year-old college student who presented to the emergency room at an outside hospital with general malaise, low-grade fever, and purplish discoloration on his face. The facial discoloration developed rapidly during the time from when he left his house to the time he arrived at the emergency room. Blood cultures were drawn and he was admitted to the intensive care unit. He was begun on imipenem-cilastatin and given fresh frozen plasma, cryoprecipitate, fluid resuscitation and dopamine. He was transferred to the University of Pittsburgh Medical Center with fever, disseminated intravascular coagulation and hypotension. Final Diagnosis -- Meningococcemia and Disseminated Intravascular Coagulation (Fever, Purpura and Hypotension)
	.

Link to image source

LABORATORY FINDINGS Peripheral Blood Decreased platelets (<20,000/ul in the acute form;  10,000/ul - 80,000/ul in the chronic form) - thrombocytopenia is due to shortened platelet survival (1-2 days versus 10 days).  Platelets are coated by an IgG antibody and removed from the circulation, primarily by the spleen but also by the liver and other reticuloendothelial organs. Large platelets are seen on smear - the early release of large immature platelets and megakaryocytic fragments from the bone marrow reflect an increased rate of platelet production. Bone Marrow Increased numbers of megakaryocytes that are less granular, smoother in contour, and more basophilic than normal megakaryocytes;  immature large megakaryocytes with increased numbers of nuclei;  absence of attached platelets indicate rapid release from the marrow Other Laboratory Findings Increased bleeding time and impaired clot retraction - reflect effect of thrombocytopenia Findings due to bleeding;  anaemia, reticulocytosis, leukocytosis

The human immune system, which protects us from disease, is made up of a complex network of highly specialized cells and organs. When any part of this network is faulty, it interrupts the smooth functioning of the immune response and can result in an immulogic disorder. Chronic granulomatous disease (CGD) is actually a group of rare, inherited disorders of the immune system that are caused by defects in the immune system cells called phagocytes. These defects leave patients vulnerable to severe recurrent bacterial and fungal infections and chronic inflammatory conditions such as gingivitis (swollen inflamed gums), enlarged lymph glands, or tumor-like masses called granulomas. While not malignant, granulomas can cause serious problems by obstructing passage of food through the esophagus, stomach, and intestines as well as blocking urine flow from the kidneys and bladder.

CGD is an inherited disease, which means that one or both parents pass on a defective gene to their child, even though the parents may be perfectly healhty. In the case of CGD, the genetic defect interferes with the child's ability to fight off certain diseases. It is estimated that one in one million babies is born with CGD, although symptoms of the disorder may not appear until after 3 months of age.

Two-thirds of people with CGD inherit the disease as a sex-linked characteristic, that is, as a defect in a recessive gene found on the X chromosome passed on to a child by its mother. A male has one X chromosome, a female has two. If a woman carries the faulty geneon only one of her X chromosome, she may not be affected by the disorder if normal phagocytes are generated by the other X chromosome. Her sons will have a 50 percent chance of inheriting the defective gene. her daughters might be carriers of the defective gene but won't develop CGD. However, carriers of the CGD gene appear to be prone to mouth ulcers and certain skin rashes.

Sometimes the normal X chromosomes in a female CGD carrier becomes inactivated early in the development of the embryo. This allows some cases of X-linked CGD to occur in females, aof X-linked CGD to occur in females, although it is much rarer than in males.

Faulty genes lodged on chromosomes other than the X chromosome can also cause CGD. The autosomal recessive form of CGD results when an abnormal gene is inherited from each parent. Both males and females are affected equally by this form of CGD. If only one abnormal gene is present, then the person carries the gene but does not develop the disease. Reference to source document Transposed from:
Chronic Granulomatous Disease
"A Guide for CGD Patients and Their Families"

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service
National Institute of Health

National Institute of Allergy and Infectonal Institute of Allergy and Infectious Diseases
Division of Intramural Research
Prepared by the NIAID
Office of Communications

Link to source

LABORATORY FINDINGS

Peripheral Blood

• Increased Hb, haematocrit, and RBC;  these parameters are markedly increased in congenital heart disease with right-to-left shunt
• Increased reticulocyte count
• *Normal WBC and platelet counts

• Normoblastic hyperplasia
• *No incrase in granulocyte precursors or megakaryocytes
• *Normal stainable iron

• *Decreased arterial oxygen saturation (<92%) - appropriate type
• Normal arterial oxygen saturation (92%) - inappropriate type
• *Normal leukocyte alkaline phosphatase
• *Increased serum erythropoietin
• *Normal serum unbound vitamin 12 binding capacity
• Normal serum iron
• Increased serum uric acid
• Increased RBC mass (total number of circulating RBC)

*These findings help in differentiating seconary erythrocytosis from polycythemia vera.

Myelofibrosis With Myeloid Metaplasia
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LABORATORY FINDINGS

Peripheral Blood*

• Normocytic anaemia, which becomes more severe as the disease progresses; microcytic anaemia occurs if the patient has had considerble bleeding.  The anaemia reflects the combined effects of increased plasma volume, reduced RBC survival and ineffective erythropoiesis.
• Marked poikilocytosis, anisocytosis, polychromatophilia, "teardrop" cells, and nucleated RBC are common findings - these reflect impaired erythropoiesis
• Slightly increased reticulocytes (<10%)
• Mild or moderate leukocytosis (<30,000/ul);  immature granulocytic elements.
• This reflects ineffective granulocytopoiesis.
• Platelet count is increased early, but decreased later;  giant and bizarrely shaped platelets and megakaryocyte fragments reflect impaired platelet formation.

• Aspirate frequently results in a "dry tap"; biopsy is necessary.
• Early, the marrow hypercellular with hyperplasia of all cell types;  later, the marrow becomes progressively less cellular and more fibrotic.
• A special stain of the marrow biopsy for reticulin shows an increase in reticulin fibres;  this is diagnostic of myelofibrosis.  These fibres are later replaced by collagen fibres.
• Megakaryocytes are numerous, often occur in clusters, frequently are abnormal in size and shape, and are the last haematopoietic element to disappear.
• Increased stainable iron reflects haemolysis due to hypersplenism or is the consequence of multiple transfusions for unresponsive anaemia.

• Elevated leukocyte alkaline phosphatase (about 67% of patients);  the level tends to fall as the disease progresses
• Increased serum uric acid reflects leukocytosis and WBC breakdown.
• Increased unconjugated (indrect) bilirubin reflects increased RBC destruction.
• Increased LDH reflects ineffective haematopoietic cellular destruction within the bone marrow.
• Moderate increase in serum vitamin B12 and in unsaturated B12 binding capacity
• Needle biopsy of spleen or liver shows evidence of haematopoiesis.

*The combination in the peripheral blood of immature RBC, WBC, and platelets is known as leukoerythroblastosis and is characteristic of myelofibrosis with myeloid metaplasia.  These morphologic abnormalities may be a consequence of damage to the normal cellular release system of the marrow, of extramedullary blood-cell formation, or of both.

Click here for link to source

Reticulin Stain

Polycythemia Vera
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LABORATORY FINDINGS

Peripheral Blood

• Increased haematocrit (60%); RBC (7 million/ul); and Hb (18 g/dl - 24 g/dl)
• Normal RBC indices
• Increased WBC (12,000/ul - 25,000/ul)
• Increased platelets (450,000/ul - 1 million/ul)
• Early in the disease, the blood cell morphology is usually normal.  Later, the blood smear shows nucleated RBC, anisocytosis, poikilocytosis, "teardrop" RBC, polychromatophilia, immature WBC, bizarre and clumped platelets.  As the disease progresses, these findings become more striking, reflecting increasing extramedullary haematopoiesis.

• Increased cellularity of all elements;  the megakaryocytic increase is prominent;  no stainable iron;  normal M-E ratio - the absence of marrow iron is characteristic, reflecting increased iron uptake, utilisation and release

• Increased RBC mass, marked - this is the most characteristic finding and is essential for diagnosis.
• Increased serum uric acid - result of increased formation of uric acid from metabolised haematopoietic nucleoprotein
• Increased leukocyte alkaline phosphatase - occurs in 80% of patients
• Increased serum vitamin B12 or unsaturated B12 binding capacity - the latter increase is greater and more characteristic than the former
• Normal arterial oxygen saturation (>92%) - this is essential for diagnosis
• Poor clot retraction
• Decreased sedimentation rate reflects increased haematocrit and blood viscosity.
• Absent or reduced serum erythropoietin;  may not be decreased when the haemoglobin is only moderately increased.

Category A

1.    Increased RBC mass
2.    Normal arterial O2 saturation
3.    Splenomegaly

Category B

1.    Thrombocytosis
2.    Leukocytosis
3.    Elevated leukocyte alkaline phosphatase score
4.    Elevated serum vitamin B12 or unsaturated B12 binding capacity

Click on image for link to source Hypercellular marrow, with both erythroid and myeloid hyperplasia in this section (Giemsa, 400x)

Essential Thrombocythaemia

LABORATORY FINDINGS

Peripheral Blood*

• Increased platelet count (> 1 million/ul) - many platelet clumps;  large and abnormal platelets; megakaryocyte fragments may be seen.
• Increased WBC (<40,000/ul) - mature granulocytes occur
• Microcytic, hypochromic anaemia - reflects bleeding

• Increase in all cellular elements, but especially in megakaryocytes, which are large and bizarre in shape
• Decreased stainable iron
• Mild to moderate increase in reticulin fibres are seen in a bone-marrow biopsy

• Abnormal platelet aggregation or adhesiveness reflects impaired platelet function.
• Increased leukocyte alkaline phosphatase
• Increased serum uric acid and vitamin B12 - reflect leukocytosis and increased WBC metabolism
• Increased serum potassium reflects increased platelet breakdown

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