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                                              Labdx test 1
                                                                                                       9/27
-under age 12, alkaline phosphatase levels are normal under 300, otherwise 30-115 is normal
-you can’t eat your glucose much above 200 unless you’re a diabetic
-confirm diabetes with FBG above 126 on 2 or more occasions
-onset of type I diabetes usually occurs under age 12
-suddenness of onset is very acute with type I
-high osmolarity from high glucose levels pulls water out of cells and into urine – polyuria
-polyuria and polydypsia are consequences of hyperosmolarity
-onset of type I diabetes: 70-80% of kids have antecedent history of flu/cold (up to 6 months prior)
    -IgM anticoxakie B virus (as opposed to IgG in normal people)
    -also will find antibodies to own Beta cells




                                           the guy with the bow tie
                                                                                                                             2
                                                       Labdx test 2                              10/15/07
4 Types of Clinical Laboratories
    -commercial/reference
    -Hospital
    -Clinic
    -Office (physician’s office lab)


Commercial / Reference Laboratories
    -A commercial lab is usually a privately owned, decentralized facility which performs lab test for private physicians,
nursing homes, out-patient health centers, and which serves as a reference lab for smaller hospitals
    -may be local, regional or national
    -purpose is profit (they sell service and good work)
    -potential      advantages:
         -full service (will order any test that is needed)
              -if they don’t provide a certain test, either nobody ever orders that test or…
         -cost
         -quality (in general, they do really good work)
         -supplies (doctor doesn’t have to buy supplies)
         -information source
         -continuing education (an educated consumer is the best customer)
              -they send you reprints of current literature
    -potential disadvantages:
         -availability (lab may be 100 miles away)
         -inconvenience
         -cost
         -quality (possible fraud)
         -turn around time (time it takes to order test and get results; from hours to months)


Selecting a commercial lab
    -doctors often buy lab services with far less scrutiny that they would use to buy a car
    -key points to think about:
        -Do they actually want your business?
             -sometimes commercial labs don’t do tests for chiropractors
         -Service
            -Do they offer all the tests you require?
            -What are the specimen/results logistics?
            -What is the usual turn around time?
                                                                                                                   10/15/07[2]
         -Price
              -How does the price per test compare?
              -Are there volume discounts?
         -Is the laboratory accredited? (state licensed, Medicare, ASCP)
         -Is there ongoing quality assurance programs? (yes, if they’re accredited)
         -Do they offer professional discounts?
         -Do they offer billing options?
         -What is the repeat testing policy?
         -Do they offer customized profiles?
         -Do they furnish any/ all supplies?
         -Are there consultation services?




                                                     the guy with the bow tie
                                                                                                                                  3
                                                                                                                         10/22/07
Hospital Laboratories
    -A centralized facility that primarily performs tests on in-patients and serves the needs of the medical staff.
    Advantages:
         Increased accessibility, quality, info source
    Disadvantages:
         -Lack of interest in taking care of your patients
         -Limited testing
         -Patient-sample-results logistics
         -cost

Clinic Laboratories
    -a centralized facility which performs tests on patients while they are at the Doctor’s office
    -Clinic labs are a type of shared facility, which is utilized by the doctors within the clinic
    -Clinic labs have essentially the same advantages and disadvantages as the hospital lab for the referring doc
    -however, unlike the commercial or hospital labs, clinic labs may be a potential source of revenue for the clinic physician


Office Laboratories
    -An office lab is a facility that is in the doctor’s office and usually performs tests only on the patients of that doctor
    -doctor’s office lab may be nothing more than a small table with a bottle of urine dipsticks or it may be an entire room
        with a chemistry analyzer, automated blood cell counter, microscopes, incubators, centrifuges, blood drawing chairs,
        and a registered Medical Technologist
     Advantages:
         -Convenience
         -Control of quality
         -Decreased cost
          -Potential source of revenue
          -Improved patient care
     Disadvantages:
         -Time and space restrictions
         -Capital expenses
         -Increased cost ?
         -Lack of quality ? (doctors don’t know what they’re doing)
         -Prohibitive regulations ?

The decision as to whether or not, to use laboratory tests in your practice is in part based on personal philosophy and in part
based on legal considerations.

The decision as to whether or not to perform laboratory test in your office should be based on practicality and cost.

There are many factors that must be considered in the evaluation of test practicality and cost, and these factors will differ for
each type of practice and practitioner.

In office testing
    In general there are a few laboratory tests that are appropriate for most out-patient based office practices.

Urinanalysis
    • pH              • Sp. gravity
    • glucose         • bile/bilirubin
    • protein         • blood
    • ketones         • nitrates
    (microscopic exam of urine sediment)
Stool exam for occult blood
Blood glucose
Culture collection supplies for throat and urine cultures
Blood drawing supplies for tests that are sent out
                                                         the guy with the bow tie
                                                                                                                            4
Do Urinalysis if patient…
   -has symptoms suggestive of possible kidney/urinary tract disorder (ie dysuria, frequency, urgency, etc)
   -exhibits signs of UT disorder such as hematuria, discoloration, urine, edema
   -has been recently treated for a UT disorder
   -has a know condition to effect the kidneys (ie HTN)
   -is undergoing treatment with a medication know to affect kidneys
   -has sustained trauma affecting GU system
   -has unexplained fever
   -is pregnant and a urinalysis is performed as part of the prenatal care if appropriate

Serology
    pregnancy test
    mononucleosis screen
    rheumatoid factor screen
    lupus/ANA screen
    rheumatic fever/ ASO screen
                                                                                                                   10/23/07[1]
Screening
    -general term that indicates the performance of a variety of tests on healthy or sick people

Criteria for Effective Screening
    -technically reliable, cost effective
    -is there an effective therapy for what you’re screening
    -at risk population must be identified
    -represent a significant health problem

Multiphasic Screening
    -groups of tests performed on healthy subjects for baseline data, risk assessment, or pre-admission (to hospitals)
    -split opinion as to the value of this screening (b/n physicians and insurance companies)
    -the more tests ordered, the more false positives found

Targeted Screening
    -Designed to confirm or exclude a specific condition
    -organ profiles
    -4-8 tests per profile
    -considered to be cost effective

-5 LDH isoenzymes
    -LDH 2 is the highest
    -if LDH 5 is high, indicates RBC or liver issue
-protein electrophoresis
    -albumin, alpha1, alpha2, beta, and gamma globulins
    -gamma globulins are made up of 5 immunoglobulins
    -“M-spike” is when gamma spikes on the electrophoresis graph (Multiple Myeloma)
    -Bence-Jones proteins in urine are light chains
                                                                                                                   10/23/07[2]
-important to know type of Hgb if anemic, otherwise it is not important
-if find protein in urine, then two things we need to know:
      what type of protein and how much is there?
          -electrophoresis reveals the type
          -24 hour urine protein to determine how much
          -albumin is the protein most associated with kidney disease
          -if multiple myeloma, then no albumin, but rather gamma-globulin spike (M-spike)
          -if no protein, electrophoresis would be a straight flat-line




                                                    the guy with the bow tie
                                                                                                                             5
Targeted screenings
    -renal panel, diabetic panel, bone/joint panel, anemia panel, etc
    -performed on someone who has a related dysfunction

Renal Panel
    -BUN/creatinine
         -dehydration inflates BUN (but not creatinine)
    -creatinine clearance
         -the semi-gold standard for kidney function (esp. glomerular filtration rate)
         -ml of serum that is completely cleared of creatinine per minute
    -total protein (serum & urine)
    -electrolytes (Na, K, Cl)
    -glucose

Pancreatic Panel
    Amylase
    Lipase – more sensitive to longer term pancreatitis
    Calcium – 55% bound and the rest available for biological function
    Glucose

Diabetic Panel
     Glucose-fasting
     Glucose 2-hr
     BUN/creatinine
     Cholesterol trigs – if high sugar, this is also high
     Glycosylated hemoglobin (measure of glycemic control) – not diagnostic for diabetes
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                                                                                                                       10/29/07

CBC
One of the most commonly ordered clinical laboratory tests, the complete blood count (CBC), is a basic evaluation of the
cells (red blood cells, white blood cells, and platelets) suspended in the liquid part of the blood (plasma).

The CBC is a useful screening and diagnostic test that is often done as part of a routine physical examination.

It can provide valuable information about the blood and blood-forming tissues (especially the bone marrow), as well as other
body systems.

 Abnormal results can indicate the presence of a variety of conditions-including anemias, leukemias, and infections-
sometimes before the patient experiences symptoms of the disease.

A complete blood count is actually a series of tests

    number of red blood cells
    number white blood cells
    number of platelets
    hemoglobin
    hematocrit (packed cell volume)
    RBC indices and morphology
    WBC differential (types & percentages)

Components of the complete blood count (hemoglobin, hematocrit, white blood cells, platelets, etc.) can also be tested
separately, and are sometimes done to monitor a specific condition

The blood count is performed relatively inexpensively and quickly.



                                                       the guy with the bow tie
                                                                                                                              6
Most laboratories routinely use some type of automated equipment to dilute the blood, sample a measured volume of the
diluted suspension, and count the cells in that volume.

RBC Count
The number of RBC's per unit volume is measured directly and given in millions per microliter.

Hemoglobin (Hgb)
The Hgb content is measured directly and given in grams per deciliter. This value, along with Hct, provides the most useful
measure of the oxygen carrying capacity of the blood.

Hematocrit (Hct)
The packed cell volume expressed as % of RBC per volume of whole blood
OR
The Hct is a calculated value (RBC count x MCV) and provides a measure of the oxygen carrying capacity of the blood.




-usually hgb is 1/3 the hct

Hemoglobin (Hgb) concentration corresponds closely to the RBC count.

 Also closely tied to the RBC and hemoglobin values is the hematocrit (Hct), which measures the percentage of red blood
cells in the total blood volume.

The hematocrit (expressed as percentage) is normally about three times the hemoglobin concentration (reported as grams per
deciliter).


Red Cell Indices
Mean Corpuscular Volume (MCV)
The MCV is measured directly; the unit is a femtoliter. The MCV measures the size of RBC's and is the most important index
for classification of anemias into "macrocytic" with higher than normal MCV and "microcytic" with low MCV.

Mean Corpuscular Hemoglobin (MCH)
The MCH is calculated (Hgb ÷ RBC count) and gives the average mass of Hgb in an individual RBC; the unit is a picogram.

Mean Corpuscular Hemoglobin (MCHC)
The MCHC is calculated (Hgb ÷ Hct)

MCV 82-98 fl (femtoliters  10-15)
MCHC 31-37 g/dl
MCH 26-34 pg (picograms  10-12)
RDW 11.5-14.5% (The RDW is the coefficient of variation of the MCV)

                                                   the guy with the bow tie
                                                                                                                                7
Red blood cell indices are useful in differentiating types of anemias.

most common causes of macrocytic anemia (high MCV):
    vitamin B12 and folic acid deficiencies

most common causes of microcytic anemia (low MCV):
    iron deficit, thalassemia, and chronic illness

most common causes of normocytic anemia (normal MCV):
    kidney and liver disease, bone marrow disorders, or excessive bleeding or hemolysis of the RBC’s

RDW is increased in anemias caused by deficiencies of iron, vitamin B12, or folic acid.

Abnormal hemoglobins, such as in sickle cell anemia, can change the shape of red blood cells as well as cause them to
hemolyze.

The abnormal shape and the cell fragments resulting from hemolysis increase the RDW.

Conditions that cause more immature cells to be released into the bloodstream, such as severe blood loss, will increase the
RDW. The larger size of immature cells creates a distinct size variation.

A platelet count is a diagnostic test that determines the number of platelets in the patient's blood.

Platelets, which are also called thrombocytes, are small disk-shaped blood cells produced in the bone marrow and involved in
the process of blood clotting.

There are normally between 150,000-450,000 platelets in each microliter of blood.

Low platelet counts or abnormally shaped platelets are associated with bleeding disorders.
High platelet counts sometimes indicate disorders of the bone marrow.

The primary functions of platelet counts are to assist in the diagnosis of bleeding disorders and to monitor patients who are
being treated for any disease involving bone marrow failure.

         Leukemia
         polycythemia vera
         aplastic anemia

An abnormally low platelet level (thrombocytopenia) may result from:
    -increased destruction of platelets
         idiopathic thrombocytopenic purpura (ITP)
         Hypersplenism
    -decreased production
         Aplastic anemia (no RBC’s, no granulocytes, no platelets)
    -increased usage of platelets
         Disseminated intravascular coagulation (DIC)

Abnormally high platelet levels (thrombocytosis) may indicate benign reaction to an infection, surgery, or certain
medications
polycythemia vera, in which the bone marrow produces too many platelets too quickly.


White Blood Cells
Leukocytes
Normal WBC = 4,500 - 11,000/cmm
Leukocytosis = an increased number of WBC
Leukopenia = a decreased number of WBC


                                                      the guy with the bow tie
                                                                                                                        8
                                                       Leukocytes


                                                        Stem Cell


  Lymphocytic               Monocytic                                            Myelocytic
                                                                               (Granulocytic)


  Lymphoblast               Monoblast             Eosinophilic                  Basophilic          Neutrophilic


 Prolymphocyte            Promonocyte                   Myeloblast                  Myeloblast          Myeloblast

                                                      Promyelocyte                Promyelocyte        Promyelocyte
   Lymphocyte               Monocyte
                                                        Myelocyte                   Myelocyte           Myelocyte

                                                     Metamyelocyte                Metamyelocyte       Metamyelocyte

                                                            Band                       Band                Band

                                                        Segmented                   Segmented           Segmented


-eosinophils – stain red/pink
-basophils – granules stain blue/purple
-neutrophils – neither pink nor blue
                                                                                                                    10/30
Case impression:
-petechiae and large bruise on back
     -no RBC’s, no WBC’s and no platelets
     -infections due to lack of WBC’s
     -bruises due to lack of platelets
     -fatigued due to low RBC’s
     -isoniazide is a hepatotoxin and marrow toxin, causing marrow suppression
          -drug-induced agranulocytosis (marrow toxicity)
-in most anemias, the WBC’s and platelets are not affected
                                                                                                                   11/1/07
RBC loses its nucleus as it matures, but retains a reticulo-stroma
-proteins stain pink, and nuclear material stains blue
-normally, 0.5-1.5% of RBC’s are reticulocytes (slightly immature RBC)
-reticulocyte is the lab test value used to make decision about hypo vs hyperproliferative anemia
-anemia: either losing too many RBC’s or not making enough
     -can tell by reticulocyte count
     -if slow bleed, then reticulocyte count is increased
     -if not making enough RBC’s, reticulocyte count is decreased (?)

White Cell Differential
-Diagnosis of myeloproliferative disorders, myelodysplasias, various other hematologic disorders
-Support diagnosis of various infections and inflammation


White Cell Differential
A determination of the relative (%) proportion of the different types of WBC



                                                    the guy with the bow tie
                                                                                                                           9
Normal Adult WBC Differential
Segs         50-75%
Bands        0-7%
Meta         0-1%
Myelo        0%
Eos          0-4%
Baso         0-2%
Lymph        20-40%
Mono         0-5%


Differential Count vs Absolute Count
-The differential count is a relative count (%), while the absolute is the actual number of cells/cmm of blood
-Absolute = % cells x the total WBC
-thus, Lymphocytosis, Lymphopenia, Lymphocytopenia, Neutrophilia, Neutropenia, Monocytosis, Eosinophilia,
Basophilia … all refer to the absolute counts NOT the differential

Leukocytosis affects all types of white blood cells:
         neutrophilia, lymphocytosis, and granulocytosis
    -leukocytosis is normally when there is a disproportionate elevation of one of the cell types

-eosinophils & allergies
     -IgE binds mast cell and causes degranulation releasing histamine and ECF, which recruits eosinophils

Normal white blood cell counts are 4,300-10,800 white blood cells per microliter.

Leukocyte or white blood cell levels are considered elevated when they are between 12,000-20,000 per microliter.

The most common and important causes of leukocytosis is inflammation and infection

    -most infections cause neutrophilia.

Tissue damage from other causes:
    burns
    infarction
    crush injuries
    inflammatory diseases
    poisonings
    severe diseases -kidney failure and diabetic ketoacidosis

-correlation (in lab testing) – if know one thing then know another thing
     -ie good correlation b/n the presence of an embryo and levels of HCG
     -good correlation b/n ALT and liver cell necrosis
     -correlations are quantifiable (via a coefficient)
     -never implies cause and effect
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                                                                                                                     11/5/07

Diseases of the RBC, WBC, and Hemostasis
RBC morphology

If RBC’s are smaller than normal and have an increases zone of central pallor. This is indicative of a hypochromic (less
Hgb in each RBC) microcytic (smaller size) anemia. There is also increased anisocytosis (variation in size) and
poikilocytosis (variation in shape)




                                                    the guy with the bow tie
                                                                                                                            10
Basophilic stippling in nucleated RBC
RBC’s have stacked together in long chains. This is known as “rouleaux formation” and it happens with increased serum
proteins, particularly fibrinogen and globulins. Such long chains of RBC’s sediment more readily. This is the mechanism for
the sedimentation rate, which increases non-specifically with inflammation and increased “acute phase” serum proteins.

    -rouleaux formation does not happen in vitro. Any kind of movement or vibration will cause the cells to dissipate apart
    -indicative of a plasma protein issue

“helmet cells” aka “schistocytes”
    -indicate the RBC’s are being destroyed (microangiopathic hemolytic anemia – MAHA)
    -typical for disseminated intravascular coagulopathy (DIC)

Features common to all types of anemia are mainly related to chronic tissue hypoxia
    -air hunger, easy fatigability, and pale skin and mucous membranes
    -Brittle and concave nails (spooning), especially in iron deficiency anemia
    -fatty change of parenchymatous organs (heart, liver)
    -expansion of red cell marrow associated with accelerated formation of RBCs

Anemias are classified etiologically according to:
   (1) Increased blood loss, due to either hemorrhage or hemolysis (hemolytic anemias)
   (2) Decreased red cell production, due to either bone marrow failure (aplastic anemia) or deficiency of important
       factors (ie iron, B12, folate)

-half life of RBCs = 120 days

Hemorrhagic Anemia
   -Acute post-hemorrhagic anemia
   -chronic blood loss anemia
   -hemolytic anemias

Acute posthemorrhagic anemia
    Manifestation of hypovolemia, hypotension, shock, and tachycardia are present in the first few hours following acute
blood loss. Later, a compensatory increase in the plasma volume occurs (hemodilution). The bone marrow actively
compensates for the loss, and the reticulocyte count increases

Chronic blood loss anemia
    It leads to iron deficiency anemia when the iron stores are depleted. It can be due to gynecologic problems with vaginal
bleeding or gastrointestinal bleeding (ie peptic ulcer or colon cancer)

Hemolytic anemia
   Hemolytic anemias have three main features common to all of them:
       -reduction in survival time of the erythrocytes
       -retention of the destroyed erythrocytes and their iron within the body
       -functionally active bone marrow

General manifestations of hemolysis
   -hyperbilirubinemia leads to jaundice
   -pigmented gallstones may form
   -intravascular hemolysis leads to hemoglobinemia with consequent hemoglobinuria
   -decreased serum haptoglobin – a hemoglobin binding serum protein found
   -excessive iron deposition leads to systemic hemosiderosis
   -splenomegaly and hepatomegaly occur as the destroyed red cells are carried out by the reticuloendothelial system
   -incr’d erythropoietic activity leads to expansion of the bone marrow. This results in thickening of the flat bones (skull)




                                                    the guy with the bow tie
                                                                                                                          11
Types of hemolysis
    -Intravascular hemolysis, which occurs when hemolysis takes place inside blood vessels owing to mechanical injury
(microangiopathic hemolytic anemia), or to a complement-mediated mechanism (mismatched blood transfusion)
    -Extravascular hemolysis, which occurs in the mononuclear phagocyte cells of the liver and spleen. This can be due to
opsonizaiton of the red cells or a red cell membrane defect. It differs from intravascular hemolysis by the lack of
hemoglobinemia and hemoglobinuria


Intrinsic (intracorpuscular) hemolytic anemia
    -hereditary spherocytosis
    -Glucose-6-phosphate dehydrogenase (G6PD) deficiency
    -Hemoglobinopathies
    -Thalassemias

I. Hereditary spherocytosis
    -It is an autosomal dominant disorder characterized by sphere-shaped red cells (spherocytes)
    -It is due to deficiency of the red cell membrane protein termed spectrin.
          This results in an unstable, deficient cell membrane. The red cells acquire a spheroidal shape.
    -Spherocytes are trapped and sequestered in the spleen with consequent splenomegaly.
          The spleen shows congested red pulp and erythrophagocytosis
    -Clinically, it manifests with microcytic anemia, splenomegaly, and hemolytic jaundice
    -infection may precipitate a hemolytic crisis or an aplastic crisis
    -characteristically, there is increased red cell osmotic fragility

II. Glucose-6-phosphate dehydrogenase (G6PD) deficiency
    This is an X-linked condition affecting 10% of African-Americans and is also commonly seen in persons of
Mediterranean origin.
    Oxidative stimuli (antimalarial drugs, infection, or fava beans) oxidize hemoglobin, creating denatured hemoglobin that
precipitates in erythrocytes as Heinz bodies.
    The latter attach to the cell membrane, decreasing its flexibility and predisposing the red cells to destruction in the
spleen.
    Clinically, it manifests with acute mild hemolytic anemia.
    G6PD deficiency protects individuals from falciparum malaria.

III. Hemoglobinopathies
These are a group of hemolytic anemias resulting from genetically determined hemoglobin (Hb) structure abnormalities

The normal hemoglobin molecule consists of two parts:
    -Heme moiety, which is the pigment containing iron (porphyrin with four pyrole rings).
    -Globin moiety, which is a protein with two pairs of polypeptide chains.

The heme moiety
    Its iron is present in the ferrous state, which is essential to oxygen transport.
    Hemoglobin oxidation into the ferric state renders it unable to carry oxygen.
          This occurs in methemoglobinemia (due to some drugs such as sulfonamides, nitrites, and phenacetin).
    Sulfohemoglobinemia occurs when sulfides combine with hemoglobin in inorganic sulfide poisoning.

The globin moiety
     There are many types of polypeptide chains that can be present in the globin moiety alpha, beta, gamma, delta, and
epsilon, thus a number of types of hemoglobin

Normal types of hemoglobin
    Hb A (adult hemoglobin) has two alpha chains and two beta chains
    Hb A2 accounts for less than 3% of normal adult hemoglobin. It has two alpha chains and two delta chains (a282)
    Hb F (fetal hemoglobin) is the normal hemoglobin in the fetus and newborn.
        It has two alpha chains and two gamma chains (cx2y2).



                                                    the guy with the bow tie
                                                                                                                             12
Hemoglobin S disorders
     This is a hereditary hemoglobinopathy resulting from a point mutation of the globin gene with consequent substitution of
valine for glutamic acid at the sixth codon of the 3 globin chain.
     This creates hemoglobin S
     It is present in approximately 7% of African Americans.
     It protects against malarial infection.
     Deoxygenation polymerizes hemoglobin S, forming rigid sickle cells that obstruct small vessels and that may produce
microinfarcts.
Hemoglobin S disorders - Consequences of sickling
    -Chronic hemolysis
    -Sickle cells possess rigid membranes, which facilitate their sequestration in the spleen
    -The life span of the sickle cells is markedly decreased
    -Microvascular occlusion
    -This results in widespread micro-infarction
    -In the spleen, this may result in auto-splenectomy- markedly shrunken spleen
    -Vaso-occlusive crisis: painful attacks of ischemic necrosis, often occurring in bones, lungs, liver, penis, spleen
    -Aplastic crisis is temporary bone marrow suppression caused by infection or folate deficiency.
         This leads to a fall in hemoglobin level.
    -Infection: Increased susceptibility to infection occurs, especially to Salmonella osteomyelitis.
         This is probably due to splenic fibrosis and an impaired alternate complement pathway

Hemoglobin C disorder- This is seen mainly in African Americans, Heterozygous forms are usually asymptomatic
unless co-inherited with hemoglobin S, Homozygous forms produce mild hemolytic anemia, splenomegaly, target cells, and
red cell crystals.

Hemoglobin E disorder- It is the second most prevalent form of abnormal hemoglobin worldwide and the third most
prevalent form in the United States- It resembles thalassemia trait and produces microcytosis.

Hemoglobin SC disease- It occurs as frequently as hemoglobin SS in African Americans- It gives the manifestations of
sickle cell anemia in a milder form.

The RBC's may sickle, but not as commonly as with Hemoglobin SS disease. The hemoglobin C leads to the formation of
"target" cells--RBC's that have a central reddish dot.
                                                                                                               11/6/07
IV. Thalassemias
These comprise a heterogenous group of hereditary disorders resulting from deficiency of production of one of the two globin
polypeptide chains of hemoglobin. They predominantly occur in persons of Mediterranean, Asian, or African ancestry.

Beta-thalassemias
    They are the most common type of thalassemias.
    They are due to reduced synthesis of the beta-globin chain.
    -there are excess alpha chains when reduction in beta chains
    The lack of beta chains results in free unbound alpha chains, which produce unstable aggregates with consequent
membrane damage, hemolysis, loss of potassium, and deficient DNA synthesis. This produces inefficient erythropoiesis, a
chronic hemolytic state, and marked splenomegaly.
    Bone marrow expansion leads to thinning of the cortical bone and new bone formation on the outer aspect. This may
create mongoloid facies in children when it involves the skull.
    Ineffective erythropoiesis is associated with excessive iron absorption.
    This, as well as repeated blood transfusions and hemolysis, creates iron overload and generalized hemosiderosis.

2 Types of Beta-thalassemias
(1) Thalassemia major
     Aka = Cooley's anemia, Mediterranean anemia
     Homozygous form
     It results in a severe transfusion-dependent form of hemolytic anemia. Peripheral blood smear shows microcytic
hypochromic red cells with anisocytosis (variability of red cell size), target cells, red cell stippling, and schistocytes
(fragmented red cells).


                                                      the guy with the bow tie
                                                                                                                             13
(2) Thalassemia minor
     Heterozygous form
     It is more common than thalassemia major.
     It is usually asymptomatic but may produce mild microcytic hypochromic anemia.

Alpha-thalassemias
These results from reduced synthesis of alpha-globin chains due to deletions of one or more of the four alpha-globin genes

Alpha-thalassemias-4 forms
    (1) Silent carrier-
         It is asymptomatic.
    (2) Alpha-thalassemia trait-
         It results in manifestations resembling Beta-thalassemia minor.
    (3) Hemoglobin H (Hb H) disease-
         It results from deletion of three of the alpha-globin genes.
         It leads to the formation of unstable tetramers of beta-globin (Hb H).
    (4) Hydrops fetalis
         It results from deletion of all four alpha-globin genes
         It leads to the formation of gamma-globin tetramers (Hb Bart's), which have a high affinity for oxygen and do not
    deliver it to tissues.
         It is lethal in utero.

Paroxysmal nocturnal hemoglobinuria
    It is a rare acquired intracorpuscular membrane defect that leads to a hemolytic disorder.
    It results from deficiency of a membrane protein termed decay-accelerating factor (DAF).
          This leads to increased sensitivity to complement mediated red cell lysis.
    DAF is also deficient in granulocytes and platelets.
          Consequently, these patients are predisposed to infections and thrombosis.
    The morning urine sample usually shows hemoglobinuria.


Extrinsic (extracorpuscular) hemolytic anemias
Autoimmune hemolytic anemia
(1) Warm-antibody AHA
    This is an IgG-mediated immune hemolytic anemia that is not associated with complement fixation.
    It is mostly idiopathic but can be secondary to the Lymphoproliferative tumors (Hodgkin's lymphoma, leukemias),
          autoimmune conditions (SLE)
    Drugs (alpha methyl dopa).
    Direct Coombs' test is positive, which indicates that the patient's red cells are coated with IgG.
    The IgG-coated (opsonized) red cells acquire a spheroidal shape (spherocytosis),
          owing to partial loss of their membranes in the spleen.

(2) Cold agglutinin AHA
     This an IgM-mediated immune hemolytic anemia.
     Cold-agglutinin IgM fixes complement at warmer temperatures and agglutinates red cells with the help of complement
          in peripheral cool parts of the body
     It can be acute or chronic. Chronic cold-agglutinin manifests with Raynaud's phenomenon, pallor,
          and cyanosis of peripheral body parts when the temperature drops below 30

Isoimmune hemolytic anemias (Rh &ABO incompatibility)
Hemolytic disease of the newborn (HDN) (aka erythroblastosis fetalis)
    It results from maternal alloimmunization against fetal red blood cell antigens, mainly the Rh system (if the mother is Rh
negative) or from ABO incompatibility between the mother and the fetus (if the mother has blood group 0).
    Antibodies are of the IgG type and can cross the placenta, leading to fetal hemolysis, stillbirth, and heart failure with
anasarca (hydrops fetalis).
    It can lead to ictherus gravis neonatorum, which may be complicated by kernicterus=permanent damage to the basal
ganglia and CNS structures due to deposition of unconjugated bilirubin.

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Microangiopathic hemolytic anemia
   It is a hemolytic state associated with widespread capillary thrombosis and thrombocytopenia. (a component of DIC)
   It is observed in malignant hypertension, eclampsia, and septic shock.

Toxic and infective hemolytic conditions
    Diseases such as malaria, clostridial and streptococcal infections may be associated with hemolysis.
    Chemical toxins such as lead, arsenic, and nitrobenzene may lead to hemolysis.

Anemias of Decreased Red Cell Production
-Iron deficiency               -Vitamin B12 deficiency
-Folic acid deficiency         -Anemia of chronic disease
-Marrow failure (aplastic)     -Marrow replacement (myelophthisic)
-Sideroblastic

-about 7% of people are classified as anemic, and 1% have Hgb values that produce symptoms

Iron deficiency anemia
    It is the most common type of anemia encountered clinically.
    It can be due to:
          -Increased demands- Pregnancy, Infancy and preadolescence.
          -Decreased intake (rare)- Infancy (Human milk is iron deficient, Prematurity, Malabsorption)
               -ferric sulfate is used as a preservative in numerous junk foods
    Chronic blood loss (the most common cause of iron deficiency in adults)
          It can be due to
               -gynecologic causes (menorrhagia)
               -gastrointestinal causes (colon cancer, peptic ulcer)  most silent form of blood loss
                    -patient can have 40-50% loss of Hgb and not be aware of it
               -hemorrhoids
               -parasitic infestations

    Clinical picture
        Dyspnea, pallor, and easy fatigability.
        Glossitis and alopecia.
        Koilonychia (nail spooning).
        Plummer-Vinson syndrome: Iron deficiency associated with an upper esophageal web and atrophic glossitis.
             It is associated with an increased incidence of pharyngeal carcinoma. It is more common in women.

    Laboratory findings
       Peripheral smear: Hypochromic microcytic red cells
       Decreased mean corpuscular volume (MCV) and MCHC
       Diminished red cell count, hemoglobin level, and hematocrit.
       Diminished serum iron level and increased total iron-binding capacity (TIBC).
       Decreased serum ferritin level.
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Anemia of chronic disease
    It occurs secondary to many chronic diseases such as rheumatoid arthritis and chronic renal diseases.
    It may be due to:
          Defective iron transport and reutilization.
          Defects in erythropoietin production.
          Shortened red cell survival
    It is usually normochromic normocytic; however, in chronic inflammatory conditions, it can be microcytic hypochromic.
    Administration of iron is usually not effective if the cause is not treated.

Sideroblastic anemia
    It can be hereditary or acquired owing to drug use (e.g., isoniazid) or lead ingestion.
    It is associated with bone marrow changes, including erythroid hyperplasia and ringed sideroblasts.
    It is characterized by increased serum ferritin, iron, and transferrin saturation. TIBC is decreased.
    It is considered preleukemic and may lead to acute leukemia.

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Megaloblastic anemia
     It is due to deficiency of vitamin B12 or folic acid and is associated with large megaloblasts (erythroid precursors) in the
bone marrow.
     There is impaired DNA synthesis leading to impaired red cell production and early red cell destruction in the marrow
(ineffective erythropoiesis). This creates a mild hemolytic tendency and results in mild hemosiderosis and elevated serum
lactate dehydrogenase (LDH) level.
     Nuclear maturation lags behind cytoplasmic maturation.
     There is abnormal granulopoiesis, leading to giant metamyelocytes and hypersegmented neutrophils.
     The peripheral blood smear shows macro-ovalocytes, pancytopenia, and hypersegmented neutrophils.

2 Types of megaloblastic anemia
    B-12 Deficiency
    Folate Deficiency

    Vitamin B12 deficiency-Pernicious anemia
         It is an autoimmune disease caused by chronic atrophic gastritis which is associated with achlorhydria and an
               increased incidence of gastric carcinoma.

         Antibodies
             Blocking antibody, which blocks the binding of intrinsic factor with vitamin B12
             Parietal antibody which binds to parietal cells.
             Binding antibody, which reacts with intrinsic factor or vitamin B12

         Clinical picture
             Pale yellow skin & Glossitis (atrophic glazed tongue)
             Atrophic gastritis with absent parietal cells that are replaced by mucus goblet cells (intestinalization).
             Subacute combined degeneration of the spinal cord (in 75% of cases), leading to sensory ataxia, spastic
                  paraparesis, hyperreflexia, paresthesias, and an impaired sense of position and vibration.
             Schilling test characterized by deficient vitamin B12 absorption, which is corrected by intrinsic factor
                  administration.

    Pernicious anemia-like vitamin B1 deficiency
         Causes
            Gastrectomy
            Terminal ileum disorders (Crohn's disease)
            Malabsorption syndromes (sprue and celiac disease)
            Diphyllobothrium latum, a fish tapeworm from improperly cooked freshwater fish
            Broad-spectrum antibiotics an

    Folate deficiency
         It produces megaloblastic anemia without neurologic manifestations or gastritis.

         Folate deficiency due to:
             Pregnancy
             Inadequate intake as in alcoholism or in geriatrics
             Malabsorption due to sprue or Giardia lamblia infection
             Drugs, dilantin and methotrexate (a chemotherapeutic agent that interferes with folic acid metabolism).

Aplastic anemia
    It is due to bone marrow depression. Characterized by hypocellular bone marrow = decreased erythroid, granulocytic,
          and megakaryocytic elements producing:
              Pancytopenia:
                   Anemia
                   Leukopenia
                   thrombocytopenia




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    Aplastic anemia can be due to
        1. Toxic chemicals and drugs
        2. Broadspectrum antibiotics (e.g., chloramphenicol).
        3. Irradiation.
        4. Viruses (hepatitis C and Parvoviruses).

Myelophthisic anemia
    It is a bone marrow replacement anemia (metastases).
    It results in a leukoerythroblastic reaction:
          myeloid precursors (e.g., myelocytes) and erythroid precursors (e.g., nucleated red cells) in the peripheral blood

Summary




POLYCYTHEMIA
Polycythemia denotes an increase in the red blood cell number that may be relative or absolute (primary or secondary)

Relative polycythemia
    Associated with diminished plasma volume = dehydration

Absolute polycythemia
    1. Primary (polycythemia rubra vera): A myeloproliferative syndrome characterized by marked erythrocytosis, moderate
granulocytosis and thrombocytosis, splenomegaly, and decreased erythropoietin (due to negative feedback).

    2. Secondary polycythemia: Caused by increased erythropoietin due to chronic hypoxia as in:
         -Chronic obstructive lung disease              - Cyanotic heart diseases
         -High altitudes                                - Erythropoietin secreting tumors

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BLEEDING DISORDERS
Excessive bleeding may result from
    (1) increased fragility of vessels
    (2) platelet deficiency or dysfunction
    (3) derangements in the coagulation mechanism
    (4) combinations of these

Primary Disorders of Hemostasis
    These are hemorrhagic disorders resulting from defects of initial platelet plug formation.
    They are characterized by petechial hemorrhages in skin and mucous membranes and bleeding from the gums, nose, and
        gastrointestinal tract.
    They result from increased vascular fragility or platelet dysfunction

Increased vascular fragility
    a. Senile purpura. It occurs on the dorsal aspects of the hands and is possibly due to age-dependent atrophy of the
         connective tissues supporting blood vessels.
    b. Simple purpura: It occurs mainly in the thighs of otherwise healthy women.
    c. Scurvy-vitamin C deficiency, resulting in bleeding gums and cutaneous petechiae.
    d. Ehler-Danlos syndrome-a connective tissue disorder due to collagen (or elastin) abnormalities, characterized by
         bleeding, deformities, articular hyperextensibility, and cutaneous hyperelasticity
    e. Henoch-Schönlein purpura-an allergic purpura due to leukocytoclastic vasculitis, characterized by purpuric rash,
         colicky abdominal pain, polyarthralgia, and acute glomerulonephritis
    f. Hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome): characterized by abnormal capillaries and
         venules that lead to hemorrhages of the skin and mucous membranes
    g. Meningococcemia and Rickettsial diseases: They affect the endothelium of small vessels, resulting in their rupture.

Disorders of platelets
These result from quantitative platelet dysfunction (thrombocytopenia)
    or
qualitative platelet dysfunction (thrombasthenia)

Platelet counts normally range between 150,000 to 300,000/mm3,
Below 100,000/mm3 is generally considered to constitute thrombocytopenia.
Spontaneous bleeding does not become evident until the count falls below 20,000/mm3

Thrombocytopenia can result from
    -decreased production (aplastic anemia, ineffective megakaryopoiesis [vitamin B12 or folate deficiency],
         or myelophthisic anemia)
    -decreased survival (drugs such as methyldopa and quinine, infections, and mechanical reasons)
    -sequestration (hypersplenism)
    -dilution (massive blood transfusion)

Idiopathic thrombocytopenic purpura (ITP)
    It results from immunologically mediated platelet destruction.
    In children, it is usually an acute self-limited condition, probably related to viral infections (rubella, cytomegalovirus).
    In adults, it is usually chronic and is associated with platelet autoantibodies. The platelets are destroyed in the spleen;
         however, splenomegaly is not a feature of ITP - Splenectomy may be curative in some cases.
    Chronic ITP is more common in women, especially in those having autoimmune hemolytic anemia, SLE, or lymphomas.
    Clinically, it manifests with easy bruisability; purpura, epistaxis, and sometimes hematuria or melena.

Thrombotic thrombocytopenic purpura (TIP)
    It is characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic manifestations, renal failure,
          and fever.
    It is caused by widespread hyaline microthrombi.
    It is more common in young women and is managed with corticosteroids and exchange transfusions.


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   The cause of hyaline microthrombus formation is not clear, but it may be due to:
       -Immunologic reaction to vascular endothelium
       -Platelet-aggregating protein
   Unlike DIC, activation of the coagulation cascade is not a feature of TTP

Isoimmune thrombocytopenia
   It is caused by antibodies against platelets (ABO incompatibility)
   Post-transfusion thrombocytopenia
   Occurs when PL-Al positive blood is given to a PL-Al negative patient who was previously sensitized
         (by pregnancy or transfusion). This results in antibody mediated destruction of platelets.

Other causes of thrombocytopenia
   Disseminated intravascular coagulation (DIC)
   Systemic lupus erythematosus
   Acquired immunodeficiency syndrome
   Acute leukemias
   Irradiation, drugs, and chemicals
   Hypersplenism
   Multiple transfusions

Qualitative platelet dysfunction - thrombasthenia
   Characterized by a normal platelet count and prolonged bleeding time

   Defective platelet adhesion:
   Bernard-Soulier disease, - deficiency of a platelet surface glycoprotein

   Defective platelet aggregation,
   acquired - aspirin inhibits thromboxane A2 formation
   inherited - Glanzmann's thrombasthenia, deficiency of the platelet membrane glycoproteins necessary for aggregation

Secondary Disorders of Hemostasis
   These are caused by defects or deficiency of the coagulation factors.
   Associated with large ecchymoses or hematomas, hemarthrosis, and prolonged bleeding after trauma

   Hemophilia A (classic hemophilia)
      It is an X-linked recessive trait characterized by factor VIII

   Hemophilia B (Christmas disease)
      It is an X-linked recessive disorder characterized by deficiency of factor IX.

   Acquired coagulation factor deficiency
      Vitamin K deficiency results from malabsorption syndromes

Combined Primary and Secondary Disorders of Hemostasis
   Von-Willebrand's disease
      It is an autosomal dominant disorder characterized by deficiency of von Willebrand's factor (vWF).
      VWF is synthesized by endothelial cells and megakaryocytes, and acts as a carrier for factor VIII
      Consequently, deficiency of vWF results in, deficient platelet adhesion and VIII dysfunction

Disseminated intravascular coagulation - DIC (consumption coagulopathy)
   It is characterized by
         -widespread microthrombi in the microcirculation, resulting Microvascular obstruction, leading to infarcts
         -Consumption of coagulation factors and platelets, leading to bleeding
   Microthrombus formation is probably due to Release of thromboplastic substances (tissue factor) during or from
         widespread endothelial injury.
   50% of DIC cases occur in complicated obstetric conditions such as eclampsia, amniotic fluid embolism,
         abruptio placentae, intrauterine fetal death, septic abortion, or puerperal sepsis.
   Around 33% of those with DIC have carcinomatosis.

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    Other causes of DIC include sepsis (especially Gram-negative septicemia) and major trauma.
    Hallmark finding of DIC: schistocytes (helmet cells – fragmented RBC’s)
    Manifestations of DIC
        Microangiopathic hemolytic anemia
        Hypofibrinogenemia, fibrin split products
        Dyspnea and cyanosis
        Convulsions and coma
        Oliguria and acute renal failure
        Shock
    Acute DIC is associated mainly with obstetric complications or severe hemorrhage and manifests mainly with bleeding.
    Chronic DIC is associated mainly with carcinomatosis and manifests with thrombotic complications (e.g., infarctions).
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Liver coagulopathy
    The liver synthesizes all coagulation factors except vWF (which is produced by endothelial cells and megakaryocytes).
    Consequently, hepatocellular failure is associated with prolonged PT, aPTT, and thrombin time.
    Platelet dysfunction and thrombocytopenia due to hepatocellular failure lead to a prolonged bleeding time.


Diseases of the White Blood Cells and Lymphoid Tissues
LEUKOPENIA
    Leukopenia is a decrease in the total number of circulating peripheral white blood cells. The most common form is a
decrease in the number of neutrophils (neutropenia, also called agranulocytosis).

     Agranulocytosis (Neutropenia) is a severe decrease in the number of granulocytes, predisposing the patient to serious
infections.

    Causes:
        Ineffective granulopoiesis
        Aplastic anemia (due to bone marrow suppression).
        Myelophthisic anemia.
        Acute leukemia.
        Drugs (anticancer drugs and broad-spectrum antibiotics).
        Megaloblastic anemia.
        Infections (typhoid fever and influenza).
    Other causes:
        Enhanced destruction of granulocytes
        Hypersplenism.
        Autoimmune disease (e.g., SLE, Felty's syndrome).
        Drugs (e.g., anticancer drugs and broad-spectrum antibiotics).

    The neutrophil count may be normal, but the function may be abnormal.

    The functional defect may involve chemotaxis and phagocytosis (lazy leukocyte syndrome), or it may be mixed and
involve intracellular digestion of organisms (Chronic Granulomatous Disease and Chediak-Higashi syndrome).

    Felty’s syndrome (in RA) – drop in white cell count in blood (white cell sequestration)
                                                                                                                   11/15/07
Diabetes: high blood glucose, high urine glucose and ketones
Renal failure: high BUN, potassium, and creatinine, low protein and albumin (blood), protein in urine
UTI: bacteria, WBC, leukocytes, and nitrates in urine
Liver failure: bilirubin elevated, elevated ALT and Alkaline phosphatase
Anemia: low RBC, HGB and HCT
Heart disease: cholesterol and trigs are elevated (esp in diabetic), possibly elevated LD




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LEUKOCYTOSIS
This is a reactive increase in the number of white blood cells, usually in response to inflammatory conditions

    Neutrophilia (Polymorphonuclear Leukocytosis)
    It is associated with acute forms of inflammation, especially the suppurative types.

    It may be associated with neutrophilic
    Döhle bodies, toxic granules, or cytoplasmic vacuolation.

More premature forms appear in peripheral blood (e.g., bands, metamyelocytes) and may be myelocytes (shift to the left).

Monocytosis: Increased number of monocytes occurs

    Granulomas (tuberculosis).
    Systemic lupus erythematosus.
    Rheumatoid arthritis.
    Inflammatory bowel diseases.

Eosinophilia: Increased number of eosinophils occurs (6 or more is unusual)

    Allergic conditions (bronchial asthma)
    Parasitic infestations
    Some skin diseases (psoriasis)
    Hodgkin's disease and eosinophilic leukemia

Lymphocytosis: Reactive increase in the number of lymphocytes occurs

    Chronic infections (tuberculosis, brucellosis, syphilis).
    Some viral infections (cytomegalovirus, Epstein-Barr virus, viral hepatitis)

Leukemoid reactions:
   Rare severe reactive leukocytosis that mimics chronic myeloid leukemia or chronic lymphoid leukemias

REACTIVE LYMPHADENOPATHY
Acute Reactive Lymphadenitis

    Localized forms occur in nodes draining areas of acute inflammation (cervical lymphadenitis in acute tonsillitis).
    Generalized forms occur in bacteremia and viral infections.
    Clinically, acute lymphadenitis may be a painful, tender, soft lymphadenopathy.

Granulomatous Lymphadenopathy (Chronic)
   Tuberculosis
   Sarcoidosis
   Toxoplasmosis
   Cat scratch disease: Gram negative bacteria
   Histoplasmosis
   Lymphogranuloma venereum: a chlamydial STD
   Tularemia: Francisella tularensis. It produces severe acute lymphadenopathy (with history of contact with rabbits).
   AIDS Related Lymphadenopathy




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                                                            Labdx test 3                                    11/19/07
                                                   LEUKEMIAS
Leukemias are malignant neoplasms of the hematopoietic stem cells of the bone marrow characterized by:
   -the appearance of abnormal immature white cells in the peripheral blood
   -bone marrow replacement with leukemic cells
   -widespread infiltration of the spleen, liver, and other organs by the leukemic cells

Leukemias are classified on the basis of:
    1. Type of cell series involved:
         Myeloid (myelogenous = granulocytic), lymphocytic, and monocytic.
    2. Level of peripheral white cell count:
         Leukemic (flooding into peripheral blood) and aleukemic (into the bone marrow only).
    3. Progression of the clinical course:
         Acute leukemia, characterized by immature (blast) cells in peripheral blood and a rapid fatal course, and
         Chronic leukemia, characterized by mature well differentiated leukocytes and an indolent course.

Chronic leukemias are associated with the highest white cell counts, and the circulating white cells tend to be of the mature
forms.
Acute leukemias usually have mild to moderate elevation of the white cell count, but the cells tend to be of the primitive
forms (blasts).

In some cases of lymphoma, cells flood into the blood and create a leukemic phase. In these cases, the type of leukemia
usually corresponds to the histologic picture of lymphoma.
     -lymphocytic lymphoma may be associated with lymphocytic leukemia
     -plasma cell (multiple) myeloma may give rise to plasma cell leukemia
     -acute myeloblastic (granuloblastic) leukemia may give rise to granulocytic sarcoma (chloroma).

The bony infiltrates of acute myeloblastic leukemia form tumor masses that usually occur in facial bones and are termed
chloromas (granulocytic sarcomas). The latter usually occur in facial bones and may lead to exophthalmos.

They are called chloromas because when they are freshly cut, they have a greenish color that fades rapidly upon exposure to
air

Subperiosteal infiltrates in acute leukemia result in bone pains and tenderness

The lymph nodes are generally enlarged in most forms of leukemia.
The most striking enlargement occurs in lymphoid leukemias
Lymph nodes are usually discrete, rubbery; and painless

The spleen is enlarged in most types of leukemia. The most striking enlargement is encountered in chronic myeloid leukemia.
The liver is enlarged in most types of leukemia.

Secondary changes include anemia, and thrombocytopenia with hemorrhages in skin, mucous membranes, and other sites.
Infection is common because the leukemic cells express little defensive capacity.

Acute lymphocytic leukemia (ALL)
Chronic lymphocytic leukemia (CLL)
Acute myelocytic (myeloblastic) leukemia (AML)
Chronic myelocytic leukemia (CML)

Acute leukemias
Acute leukemias are characterized by a rapidly fatal course and predominance of immature (blast) cells in the peripheral
circulation and bone marrow.
They represent the most common malignant tumor in children.
The blasts suppress the normal hematopoietic stem cells and result in pancytopenia.

Abrupt stormy onset: Most patients present within 3 months of the onset of symptoms.


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Symptoms related to depression of normal marrow function: fatigue due mainly to anemia; fever, reflecting an infection due
to absence of mature leukocytes; bleeding (petechiae, ecchymoses, epistaxis, gum bleeding) secondary to thrombocytopenia.
Acute lymphoblastic leukemia (ALL)
     It constitutes more than 80% of childhood acute leukemias.
     B cells (CD19 B-cell marker) comprises about 80% of cases of ALL
     ALL has the best response to chemotherapy among the acute leukemias

Acute myeloblastic leukemia (ANL)
   It is also termed acute granulocytic leukemia and acute nonlymphoblastic leukemia
   It most commonly affects young adults
   It shows a predominance of myeloblasts and promyelocytes.
   The cytoplasm shows fine azurophilic granules with red rodlike structures (Auer bodies)
   The prognosis is worse than for ALL

Chronic leukemias
    These are characterized by more mature forms and less blasts than in acute leukemias.
    They follow a longer course than acute leukemias and usually express remissions and exacerbations.

Chronic myeloid (myelocytic; granulocytic) leukemia (CML)
     It is a myeloproliferative disorder that affects young adults and is characterized by neoplastic clonal proliferation of
myeloid stem cells.
      Leukocyte count is markedly elevated (usually more than 100,000/mm3) with many neutrophils, metamyelocytes,
myelocytes, basophils, and eosinophils. Thrombocytosis can be encountered early in the disease.
     Approximately 90% of cases express Philadelphia chromosome
     Splenomegaly is usually severe, while hepatomegaly and generalized lymphadenopathy are mild.
     CML expresses an accelerated phase with severe anemia, thrombocytopenia, and excess blasts (blastic crisis) with
transformation into acute leukemia.
     In contrast to ALL, the presence of Philadelphia chromosome in CML carries a good prognosis.

Chronic lymphoid (lymphocytic) leukemia (CLL)
    It occurs in old age (usually older than 60), is more common in men, and runs an indolent course.
    Leukemic lymphoid cells are usually of the B-cells that are unable to differentiate into plasma cells. This leads to
hypogammaglobulinemia and predisposes the patient to bacterial infections.
    Lymphadenopathy is marked

Hairy cell leukemia
    It is a rare chronic B-cell lymphocytic leukemia occurring in old age.
    Cells have hair-like surface projections.
    It is associated with marked splenomegaly and pancytopenia (due to marrow failure and hypersplenism).
    Splenectomy may be of benefit

MYELODYSPLASTIC SYNDROMES
    It is a group of bone marrow disorders characterized by ineffective or disordered maturation and hypercellular or
          normocellular bone marrow.
    There are usually megaloblastoid erythroid precursors, hypogranular myeloid precursors, and an increased number of
          bone marrow blasts.
    The peripheral smear shows micromegakaryocytes and agranular platelets.
    Refractory anemia and Chronic myelomonocytic leukemia
    It usually affects elderly males. The median survival ranges from 1 to 15 years.

PLASMA CELL DYSCRASIAS
    These are monoclonal neoplastic proliferations of immunoglobulin-secreting plasma cells.
    They secrete monoclonal immunoglobulin, which is usually a complete immunoglobulin, light Bence Jones protein
         or heavy chains, or both.
    The light chains include lambda or kappa (Bence Jones proteins).
    If only light chains are produced (without complete immunoglobulin), the condition is referred to as light chain disease.



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MYELOPROLIFERATIVE SYNDROMES
    These are clonal neoplastic proliferations of multipotent bone marrow stem cells
    Polycythemia Rubra Vera
        -an absolute increase in the red cell mass (severe erythrocytosis).
        -moderate granulocytosis as well as thrombocytosis and splenomegaly.
        -erythropoietin level is decreased. This distinguishes it from secondary polycythemia, which is associated with
             increased erythropoietin level.
        -Clinically, in patients with polycythemia rubra vera, there is increased viscosity, thrombosis, hemorrhagic diathesis,
             vascular stasis and cyanosis.
        -Headaches, dizziness, infarcts, and hyperuricemia are also present

Multiple (plasma cell) myeloma
    It is the most common gammopathy.
    It is a malignant neoplasm of plasma cells involving multiple sites in bones.
    It produces a monoclonal immunoglobulin, termed M protein, that appears in the serum and urine on electrophotometry
          as an M spike. The total serum protein level is increased with hyperglobulinemia.
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    The M component is usually IgG, less commonly, it may be IgA, IgD, or IgE
    It may be associated with a free light chain either lambda or kappa (Bence Jones proteins), which may appear in the
          urine)

Clinical pathologic features

    Bone lesions
       1.They are due to an osteoclast-activating factor released by the tumor cells.
       2. They are multiple osteolytic punched-out (with a clearly defined sharp border) lesions involving mainly the skull
             and axial bones.
       3. Osteopenia (diffuse bony rarefaction due to demineralization) may be present.
       4. They produce pain, pathologic fracture, and hypercalcemia due to bone resorption.
       5. Hypercalcemia leads to confusion, lethargy, depression, constipation and metastatic calcification.

        Multiple myeloma presents most often as multifocal destructive bone lesions throughout the skeletal system
        vertebral column, 66%; ribs, 44%; skull, 41%;
        Pathologic fractures are most common in the vertebral column.

        Hyperglobulinemia results in hyperviscosity syndrome and rouleaux formation (stacking of RBCs in blood smear)
        Pancytopenia (incr RBCs, WBCs, and platelets) is due to bone marrow replacement by the neoplastic plasma cells.
        Recurrent encapsulated bacterial infection (pneumococcal) occurs owing to suppression of normal immunoglobulin.

        Myeloma kidney occurs due to tubular casts of Bence Jones proteins and tubular atrophy
        Renal amyloidosis
        Metastatic calcification
        Recurrent pyelonephritis (due to decreased immunity)
        It results in azotemia and renal failure.
        The most common causes of death are renal failure and infection.

Waldenstrom’s macroglobulinemia
    It is a malignant proliferation of monoclonal IgM-producing cells
    It most commonly affects elderly males.
    The M protein is an IgM of either lambda or kappa specificity.

    It produces hyperglobulinemia and
    hyperviscosity syndrome. which may lead to dizziness, headache, stupor, and retinal vasodilatation with hemorrhage.

    It is a slowly progressive condition with generalized lymphadenopathy, anemia, and hepatosplenomegaly
    The average survival rate is 2-5 years with chemotherapy



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Heavy-chain diseases
These are rare gammopathies associated with an increased level of one of the heavy chains in the blood or urine, or both.
Alpha heavy-chain disease is the most common type of heavy chain disease and occurs mainly in young adults
Gamma-chain disease, encountered most often in the elderly, resembles a malignant lymphoma
Mu-chain disease is the rarest of these entities, most often encountered in patients with chronic lymphocytic leukemia

Histiocytosis
This is a proliferative disorder of the Langerhans' cell, a phagocytic cell present normally in the epidermis and has three
variants

Letterer-Siwe Disease
    It is also termed acute disseminated Langerhans' cell histiocytosis.It affects infants and young children and has an
    aggressive fatal course. It is characterized by widespread histiocytic proliferation with hepatosplenomegaly,
    lymphadenopathy, pancytopenia, and recurrent infections.

Hand-Schüller-Christian Disease
   It is also termed multifocal Langerhans' cell histiocytosis.
   It affects children younger than the age of five and has a better prognosis than Letterer-Siwe disease.It is characterized
   by histiocytic proliferation with inflammatory cells leading to hepatosplenomegaly, lymphadenopathy, calvarial bone
   involvement, posterior pituitary involvement, diabetes insipidus, and exophthalmos.

Eosinophilic Granuloma
    It is also termed unifocal Langerhans' cell histiocytosis.
    It affects children and young adults and carries the best prognosis of all histiocytosis variants.It is characterized by many
    eosinophils and by histiocytic proliferation mixed with inflammatory cells.
    It is usually a solitary asymptomatic bone lesion


LYMPHOMAS
    Lymphomas are malignant neoplasms (monoclonal) of lymphoid tissues (lymphocytes and histiocytes)
    The term “lymphoma” is something of a misnomer, since these disorders are lethal unless controlled or eradicated
    through therapy. There are no “benign” lymphomas. Two broad categories include Hodgkin's and non-Hodgkin's
    lymphomas.

Hodgkin’s Disease
    This is a malignant lymphoid neoplasm that may occur at any age but shows two peaks of incidence: one at the third
          decade of life and the other at the seventh decade.
    It is the most common type of lymphoma and predominantly affects men

Clinical features of Hodgkin’s
    It presents with painless enlargement of the lymph nodes, which spreads to anatomically contiguous nodes
    It is uncommon for Hodgkin's disease to involve extranodal structures (liver)
    Fever (recurring fever), pruritus, diaphoresis, and leukocytosis.

    Ann Arbor classification
        1. Stage I: Disease involves a single node or a group of nodes.
        2. Stage II: Disease involves more than one group of nodes along one side of the diaphragm.
        3. Stage III: Disease involves nodes on both sides of the diaphragm.
        4. Stage IV: Widespread involvement of extralymphoid sites is seen with or without nodal involvement.

         Clinical staging is better correlated with the prognosis than is the histologic variant

    Histologic classification: The characteristic cell is the Reed-Sternberg cell, a large binucleated or multinucleated) cell
        with eosinophilic nucleoli surrounded by a clear halo (owl-eye appearance),
    The more Reed-Sternberg cells, the more aggressive the disease and the worse the prognosis




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                                                                                                                               25
Non-Hodgkin’s Lymphomas
    -These are malignant tumors of lymphoid tissues that are characterized by more frequent involvement of multiple
        peripheral nodes than in Hodgkin's disease. They commonly involve extranodal tissues.

    -The usual presentation of NHL is as a localized or generalized lymphadenopathy.
    -Although variable, all forms of lymphoma have the potential to spread from their origin in a single node or chain of
        nodes to other nodes, and eventually to disseminate to the spleen, liver, and bone marrow

Burkitt's lymphoma
    It is B cell lymphoma usually affecting children.
    It occurs sporadically in the United States, where it usually presents with an abdominal mass.
    It is endemic in some parts of Africa and usually presents with a facial bone mass.
    It is related etiologically to Epstein-Barr virus infection.

DISEASES OF THE SPLEEN
Hypersplenism
    It is associated with splenomegaly
    Blood cells are sequestered at an increased rate in the enlarged spleen, leading to pancytopenia
    Reactive hyperplasia of the bone marrow occurs
    Splenectomy leads to clinical and hematologic improvement

Hyposplenism
Lack of splenic function is encountered in:
    -Splenectomy
    -Sickle cell disease (autosplenectomy due to repeated infarctions)
    -Celiac disease (splenic atrophy

It predisposes patients to recurrent bacterial infections, especially by capsulated bacteria (Streptococcus pneumoniae,
Hemophilus influenzae, Neisseria meningitidis, and Escherichea coli).
Septicemia and multi-organ failure may develop eventually.

Splenomegaly
Causes include:
    -Infective
    -Neoplastic: Lymphomas & Leukemias, especially chronic leukemia (the spleen is largest in CAM)
    -Immunologic:
        -Sarcoidosis (noncaseating granulomas).
        -Felty's syndrome: Follicular hyperplasia of the spleen associated with hypersplenism and leukopenia in an adult
             with rheumatoid arthritis.
        -Systemic lupus erythematosus

                                                 INFLAMMATION
Inflammation IS NOT Infection. Inflammation is a reaction of the microcirculation characterized by movement of fluid and
leukocytes from the blood into the extravascular tissues. Inflammation is a process which attempts to localize and eliminate
metabolically altered cells, foreign particles, micoorganisms, or antigens. Under normal conditions the inflammatory
response eliminates the pathogenic insult and removes the injured tissue components

An inflammatory reaction or response leads to one of three possible outcomes:
    1) The source of the tissue injury is eliminated, the inflammatory response resolves, and normal tissue architecture and
           physiologic function are restored
    2) The tissue is irreversibly injured despite elimination of the initial pathogenic insult, the affected tissue’s architecture
           Is altered (scarred), and varying degrees of function are retained
    3) The inflammatory response fails to eliminate the pathologic insult, the inflammatory response continues, the tissue’s
           architecture is continually altered, and function is eventually lost
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                                                                                                                             26
Inflammations is almost always accompanied by some degree of cellular necrosis. It is the final common pathway of almost
all pathological processes. The characteristics of an inflammatory reaction are swelling, heat, redness, and pain and the
systemic presentation of inflammation includes fever, tachycardia, malaise, and anorexia.

Depending on the site of inflammation (liver, kidney, muscle) a number of laboratory tests may be abnormal, and reflect the
associated cellular necrosis
     CPK - increased due to tissue destruction
     LDH - increased due to tissue destruction
     ALT/AST - increased due to tissue destruction
Despite the location or the etiology of the inflammation one or more of the following supports the presence of an
inflammatory reaction:
     Increased WBC ( >12,00/cmm)
     WBC Left Shift (> 10% bands)
     Increased ESR (> 20 mm/hr)
     Increased C-Reactive protein(>10ug/ml)
     Increased alpha and beta globulins

LEUKOCYTOSIS
Leukocytosis can be a false positive indicator of inflammation as it occurs in a number of non-inflammatory conditions, such
as:
       Pregnancy
       Acute bleeding
       Following severe exercise
       Daily circadian fluctuation of about 2x

Additionally, some infections typically do not produce an elevation of the WBC
        Bacterial - Brucellosis, Chlamydial, Diptheria, Typhoid
        Fungal - Histoplasmosis
        Parasitic - Malaria, Toxoplasmosis, Leishmaniasis
        Rickettsial - Typhus, Rocky Mountain Spotted Fever
        Viral - Chickenpox, Measles, Rubella, Smallpox

LEFT SHIFT - An increase in the number of immature granulocytes

Immature granulocytes: Basophilic, Eosinophilic, or Neutrophilic
       Bands or stabs
       Metamyelocytes or Juvenile
       Myelocyte
       Promyelocyte

“Right Shift” - An increase in the number of mononuclear cells :     lymphocytes or monocytes
“Left Shift” - The increase number of immature cells in the peripheral circulation occurs because cells are recruited out,
before they have fully matured

Left shift = increased number of baso, eos, and neutrophils
Right shift = increased number of lymphocytes and monocytes

SED RATE - ERYTHOCYTE SEDIMENTATION RATE
When well mixed venous blood is placed in a vertical tube, the RBC will tend to fall toward the bottom. The length of fall of
the top of the column of RBC in a give time is the Erythrocyte Sedimentation Rate (ESR). ESR is usually expressed in
mm/hr.

ESR – let tube set for an hour, the sed rate is the mm distance that the top of the cells are from the original top
        -if inflammation, sed rate is elevated




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                                                                                                                          27
What is the maximum possible Sed Rate?
         First, it depends on the length of the tube
         Second , it depends on the Hematocrit
   V = 2r2 (d1- d2) g
            9n
   r = radius of the sphere
   d1= density of the sphere
   d2= density of the fluid
   g = force of gravity
   n = Viscosity of the fluid

    What’s being measured?
    How does this relate to inflammation?

Plasma Composition
RBC are negatively charged and normally repel each other.
The negative charge is expressed as the Zeta potential

The Zeta potential is a function of the:
        -sialic acid groups on the RBC membrane
        -pH of the medium
        -ionic strength of the medium
        -dielectric effect of the protein molecules in the medium.

All proteins and other macromolecules decrease the zeta potential of the RBC

During an inflammatory response acute phase proteins are produced, which contribute to lowering the zeta potential

Decreased Zeta potentials allow for RBC to undergo rouleaux formation (pseudoagglutination), thus causing the RBC to fall
at a greater rate (i.e.; to increase SEDIMENTATION rate)

Rouleaux formation (pseudoagglutination)

    The proteins that contribute the most lowering the Zeta potential are:
                   Fibrinogen
                  Gamma globulin
                   alpha 1 globulin
                   beta globulin
                   haptoglobin

    Acute Phase Protein Effect on Sed Rate
    Inflammatory response  acute phase proteins  rouleaux formation

    *Zeta potential is the net negative charge (of plasma?)
    -zeta potential allows for Rouleaux formation which promotes the sed rate
    -elevated sed rate is indicative of an increase in acute phase proteins in patients plasma


Sed Rate and “Artifacts”
There are times when Sed Rate results are meaningless and thus the Sed Rate can not be used

Artifacts: NUMBER OF ERYTHOCYTES
When the number of erythrocytes per unit volume of blood is greater or less than normal, the true sedimentation rate is
modified.
Hematocrit values greater than 42±5 for females and 47±5 for males will, by them selves, produce clinically significant
sedimentation rates.



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                                                                                                                         28
    Example:
        A patient has a Hematocrit of 30%, has no inflammatory reaction or process, yet has a Sed Rate of 36mm/hr
    This is a false positive result (an abnormal Sed Rate in the absence of inflammation) due to the decr numbers of RBC
        Conversely, a patient has a Hematocrit of 60%, has a severe inflammatory reaction or process, yet has a Sed Rate of
    5 mm/hr. This is a false negative result (an normal Sed Rate in the presence of inflammation) due to the incr numbers of
    RBC

    With Hct normally 45%,
        -an Hct of 30% would FALSELY INCREASE erythrocyte number
        -an Hct of 60% would FALSELY DECREASE erythrocyte number

Artifacts: SIZE OF ERYTHROCYTES
Macrocytosis (MCV>96) or Microcytosis (MCV<82) will produce clinically significant sedimentation rates
    EXAMPLE:
         A patient has an MCV of 70, has no inflammatory reaction or process, yet has a Sed Rate of 36mm/hr
    This is a false positive result (an abnormal Sed Rate in the absence of inflammation) due to the decreased size of RBC
         Conversely, a patient has an MCVof 105, has a severe inflammatory reaction or process, yet has a Sed Rate of 5
    mm/hr. This is a false negative result (an normal Sed Rate in the presence of inflammation) due to the decreased size of
    RBC

    If MCV is normally 90,
        -an MCV of 70% would FALSELY INCREASE erythrocyte number
        -and an MCV of 105% would FALSELY DECREASE erythrocyte number

Artifacts: SHAPE OF ERYTHROCYTES
When the shape of erythrocytes is such as to make rouleaux formation impossible, a low or falsely normal sedimentation rate
will be produced. Moderate to marked anisocytosis, acanthocytosis, spherocytosis, or sickling of RBC render the
Sedimentation rate of no value

Remember: NEVER EVALUATE A SED RATE RESULT WITHOUT KNOWING THE HCT, MVC AND RBC
MORPHOLOGY. If you can’t use or evaluate a Sed Rate, measure the Acute Phase Proteins

ACUTE PHASE PROTEINS
Acute phase proteins are nonspecific indicators of inflammation

The clinically useful acute phase proteins are:
              C-reactive protein
              Alpha-1-antitrypsin
              Fibrinogen
              C3
              C4
              Cerulplasmin

                                Normal Acute Inflammation         Response
                                  (mg/dl)   (mg/dl)                 (hrs)
    C-reactive protein          0.08-0.4      40                   6-10
    Alpha-1-antitrypsin         200-400       700                  24
    Fibrinogen                  200-450       1000                 18-24
    C3                          55-120         300                 24
    C4                          20- 50         100                 24
    Cerulplasmin                15- 60         200                 48-72

Of the acute phase proteins, the CRP is most often used :
         -it is the most sensitive(100x rise)
         -rises the most quickly(6-10hrs)
         -is not subject to as many artifacts as the ESR




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                                                                                                                               29
Acute phase proteins are useful for:
        -screening for organic disease
        -monitoring disease activity
        -monitoring response to therapy
        -differential diagnosis
                                                                                                                         11/29/07
      Case impression:
          -do urine culture to confirm urinary tract infection
          -left shift = inflammatory/infection
          -nocturnal pain in lower legs, plus high glucose = possibly diabetic neuropathy
          -if protein in urine, need to know what kind and how much
          -if diabetic nephropathy, most likely albumin
          -if blood in urine, could be a false positive protein
          -urinary tract infection is not uncommon in a diabetic
          -people that have urinary tract infections, tend to have recurring UTI’s
                                                                                                                          12/3/07
                                                        Urinalysis
Urinalysis can reveal diseases that have gone unnoticed because they do not produce striking signs or symptoms.
    1. The patient has symptoms suggestive of possible kidney/urinary tract disorder, e.g., dysuria, frequency, hesitancy,
        nocturia, urgency, flank pain, pelvic pain, abdominal pain,
    2. The patient exhibits signs of kidney/urinary tract disorder such as hematuria, discoloration of urine, edema
    3. The patient has been recently treated or is under treatment for a urinary tract disorder, and a repeat urinalysis is
        necessary to evaluate the patient.
    4. The patient has a condition known to affect the kidneys or urinary tract , such as hypertension, diabetes mellitus,
        collagen vascular disease, etc.,
    5. The patient is undergoing treatment with a medication known to potentially adversely affect the kidneys, such as a
        chemotherapy agent.
    7. The patient has sustained trauma affecting the genitourinary system.
    8 The patient has unexplained fever.
    9 Patient is pregnant and a urinalysis is performed as part of the prenatal care if appropriate.

-The most cost-effective device used to screen urine is the dipstick.
-This microchemistry system has been available for many years and allows qualitative and semi-quantitative analysis within
one minute by simple but careful observation.
-The first part of a urinalysis is direct visual observation. The color of the urine sample is assessed subjectively and reported
as red, brown, yellow, etc, or combination thereof, along with a modifier for the depth of color, eg, light yellow, dark
red/brown, etc
-Normal, fresh urine is pale to dark yellow or amber in color and clear.

Various urine colors and common causes
light to medium yellow              normal
colorless                           very dilute urine
very dark yellow                    extremely concentrated; bilirubinuria
red to brownish red                 hematuria, hemoglobinuria, myoglobinuria
reddish brown to brown              myoglobinuria, hemoglobinuria, methemoglobin
greenish tint                       bilirubinuria
Orange                              drugs (pyridium, rifampin, others
Bright blue                         drugs (methylene blue, others
Fluorescent yellow                  vitamins
Red                                 drugs (phenolphthalein, phenothiazines), beets



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                                                                                                                               30
-Normally, fresh urine is clear to very slightly cloudy
-The turbidity of the urine sample is gauged subjectively and reported as: clear, slightly cloudy, cloudy, opaque, or flocculent
-Excess turbidity results from the presence of suspended particles in the urine
-urine pH means almost nothing

Common causes of abnormal turbidity
     increased cells (RBC, WBC)
     numerous crystals: urates/phosphates
     bacteria
     lipiduria (lipids often rise to the surface)
     mucus
     semen
pH
The glomerular filtrate of blood plasma is usually acidified by renal tubules and collecting ducts from a pH of 7.4 to about 6
in the final urine.

The pH of a urine sample is affected by a variety of factors

renal H+ excretion and HCO3- resorption
    -pathologic abnormalities of systemic acid/base balance.
    -pathologic abnormalities of tubular function: with failure to excrete an acid load or failure to absorb bicarbonate.
    -dietary factors - due to differences in dietary "acid load",
    -“herbivores” usually have alkaline urine “carnivores” tend to have acidic urine
    -Urine does become slightly less acidic after eating, associated with a post-prandial alkaline tide (due to increased
secretion
         of HCl into the stomach).

-age of specimen (loss of CO2 from the sample to the air raises the pH)
-presence of contaminant or pathogenic bacteria (some convert urea to ammonia, raising pH)
-Knowledge of the urine pH is important in interpreting urine sediment findings.
-Erythrocytes, leukocytes, and casts tend to disintegrate in alkaline urine (pH > 8.0)
-In addition, precipitation of urine crystals in supersaturated urine is highly dependent on urine pH
-Although the kidneys play a central role in the control of acid/base metabolism, the pH of a random urine sample is not a
reliable indicator of total body acid/base status.
-In some conditions, impaired renal tubular function in fact causes or perpetuates the underlying acid/base derangement.
-Meaningful evaluation of acid-base status requires blood gas analysis and consideration of clinical signs.
-The test is specific for the detection of hydronium ions, the pH being the negative common logarithm of the hydronium ion
concentration. The test pad contains the indicators methyl red, phenolphthalein and bromthymol blue.


Specific Gravity (USG)
-tells fluid intake
-Urine specific gravity is a measurement of the density of urine compared to pure water
-The USG is influenced by the number of molecules in urine, as well as their molecular weight and size, therefore it only
approximates solute concentration.
-It is also affected by temperature, with urine density decreasing with increasing temperatures.
-The presence of large amounts of protein and glucose will alter the USG & should be considered when interpreting USG
results
-Urine osmolality is directly related to the number of particles in solution and is unaffected by molecular weight and size.
-Osmolality can be measured by freezing point depression
-Urine osmolality can be approximated from the USG, by multiplying the last 2 digits of the USG by 36
-Specific gravity between 1.002 and 1.035 on a random sample should be considered normal if kidney function is normal.
- Since the USG of the glomerular filtrate in Bowman's space ranges from 1.007 to 1.010, any
measurement below this range indicates hydration and any measurement above it indicates relative dehydration.

-If USG is not > 1.022 after a 12 hour period without food or water, renal concentrating ability is impaired and the patient
either has generalized renal impairment or nephrogenic diabetes insipidus.
- In end-stage renal disease, USG tends to become 1.007 to 1.010.

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                                                                                                                                31
-Any urine having a specific gravity over 1.035 is either contaminated, contains very high levels of glucose, or the patient
may have recently received high density radiopaque dyes intravenously for radiographic studies or low molecular weight
dextran solutions.

The test detects the ion concentration of the urine. In the presence of cations, protons are released by a complexing agent and
produce a color change in the indicator bromthymol blue from blue via blue-green to yellow.
(This test is based on the apparent pKa change of certain pretreated polyelectrolytes, poly(methyl-vinyl-ether/maleic
anhydride), in relation to ionic concentration. In the presence of bromthymol blue, colors range deep blue-green in urine of
low ionic concentration through green and yellow-green in urines of increasing ionic concentration.)


Protein
Dipstick screening for protein is done on whole urine, but semi-quantitative tests for urine protein should be performed on the
supernatant of centrifuged urine since the cells suspended in normal urine can produce a falsely high estimation of protein.

-The urine protein results should always be interpreted in context with the urine specific gravity and pH.
-A trace to 1+ reaction in a very dilute urine is suggestive of significant proteinuria.
-A dipstick protein reaction > 2+ in dilute urine indicates significant proteinuria.
-Normal total protein excretion does not usually exceed 150 mg/24 hours or 10 mg/100 ml in any single specimen.
-More than 150 mg/day is defined as proteinuria.
-Proteinuria > 3.5 gm/24 hours is severe and known as nephrotic syndrome.
-The test is based on the "protein error of pH indicator dyes".
-Basically, the test is dependent on the ability of amino groups in proteins to bind to and alter the color of acid-base
indicators, even though the pH is unchanged
-The reaction is extremely sensitive to albumin (as it contains the most amino groups), but is much less sensitive to globulins.
-It is insensitive to Bence-Jones proteins.
-Generally this differential sensitivity is not a significant problem (nearly all cases of significant proteinuria involve
albuminuria)

Protein - False positive results
    Alkaline urine: False positives occur rarely in highly buffered or alkaline urine samples as the citrate buffer is
overcome, resulting in a shift in pH.
    Contact time: Leaching of the citrate buffer occurs if the urine remains in contact with the pad for a long time.
    Detergents: Quaternary ammonium compounds and chlorhexidine can result in false positives.

In rough terms: trace positive results (which represent a slightly hazy appearance in urine) are equivalent to 10 mg/100 ml
or about 150 mg/24 hours (the upper limit of normal).

1+ corresponds to about 200-500 mg/24 hours,
2+ to 0.5-1.5 gm/24 hours
3+ to 2-5 gm/24 hours,
4+ represents 7 gm/24 hours or greater.

The most accurate measurement of urine protein output is measurement of urine protein excretion over 24-hours

Things beside kidney disease can cause proteinuria:
     -Postural proteinuria (orthostatic, etc): 3-5% of healthy young adults pass excess protein during the day, not at night.
     -Functional proteinuria (albuminuria): occurs with fever, cold exposure, stress, pregnancy, eclampsia, CHF, shock,
severe exercise
     -Drugs that can increase measurements include acetazolamide, aminoglycosides, amphotericin B, cephalosporins,
colistin, griseofulvin, lithium, methicillin, nafcillin, nephrotoxic drugs (such as arsenicals, gold salts), oxacillin,
penicillamine, penicillin G, phenazopyridine, polymyxin B, salicylates, sulfonamides, tolbutamide, and viomycin.




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                                                                                                                                32
Glucose
-In nearly all cases, glucosuria is a result of prior (often, continuing) hyperglycemia to a level in excess of the renal threshold
for reabsorption
-Glycosuria generally means diabetes mellitus.
-Benign low renal glucose threshold (the kidneys excrete glucose in the urine at a relatively low blood glucose level)
-Cushing's syndrome
-Severe stress (for example, trauma or surgery)
-Drugs that may increase urine glucose measurements include: aminosalicylic acid, cephalosporins, chloral hydrate,
chloramphenicol, dextrothyroxine, diazoxide, diuretics (loop and thiazides), estrogens, isoniazid, levodopa, lithium, nafcillin,
nalidixic acid, and nicotinic acid (large doses).

-Glucose is measured on the Multistix by a glucose oxidase method. The reaction of glucose with glucose oxidase forms
nascent oxygen (O), which converts potassium iodide in the dipstick pad to iodine, forming a brown color change. Normal
urinary glucose is below the level of sensitivity of the commonly used detection techniques. Therefore, glucose is an
abnormal finding in urine.

-Drugs that may increase urine glucose measurements include: aminosalicylic acid, cephalosporins, chloral hydrate,
chloramphenicol, dextrothyroxine, diazoxide, diuretics (loop and thiazides), estrogens, isoniazid, levodopa, lithium, nafcillin,
nalidixic acid, and nicotinic acid (large doses).

False positives (trace to +1)
dipstick jar being left uncapped for a few days hypochlorite( bleach)

Drugs that may give false negative results
    ascorbic acid
    levodopa
    Phenothiazines
    Tetracycline


Ketones
Ketonuria indicates deranged energy metabolism such that fat is used in excess of carbohydrate. This can result in production
of the ketone bodies (acetone, aceotacetic acid, beta-hydroxybutyric acid) in amounts greater than can be metabolized by
peripheral tissue and filtration into urine in excess of tubular reabsorption

Test is based on the principle of Legal's test Acetone and acetoacetic acid react with sodium nitroprusside in alkaline solution
to give a violet colored complex

Phenylketones and phthalein compounds produce red colors on the test area.

 Captopril, MESNA (2-mercaptoethanesulfonic acid sodium salt) and other substances containing sulfhydryl groups may
produce false-positive results

metabolic abnormalities
   uncontrolled diabetes or glycogen storage disease

abnormal nutritional conditions
   starvation, fasting, anorexia, high protein or low carbohydrate diets

protracted vomiting

disorders of increased metabolism
    hyperthyroidism, fever, acute or severe illness, burns, pregnancy, lactation or following surgery

-Because the color change on the dipstick can be quite subtle, positive reactions can confirmed with the Acetest.
-This comes in tablet form and contains lactulose to enhance the color change.
-The Acetest is useful for semi-quantitatively measuring ketones in other fluids, such as plasma, serum and milk.



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Nitrite
-The reaction thus indirectly detects the presence of nitrite-forming organisms in the urine..
-The test is based on the principle of Griess' test and is specific for nitrite

-The most common organisms causing urinary tract infections, E. coli, and most urinary pathogens, convert dietary nitrate to
nitrite, which produces a pink coloration of the test area .

-This assay determines total nitric oxide based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. The
reaction is followed by a colorimetric detection of nitrite as an azo dye product of the Griess Reaction. The Griess Reaction is
based on the two-step diazotization reaction in which acidified NO2 produces a nitrosating agent, which reacts with sulfanilic
acid to produce the diazonium ion. This ion is then coupled toN-(1-naphthyl) ethylenediamine to form the chromophoric azo-
derivative

-Negative results do not exclude significant bacteriuria
-A negative result even in the presence of bacteriuria can have the following reasons:

-bacteria not containing nitrate reductase, diet with low nitrate content, high diuresis, high content of ascorbic acid or
insufficient incubation of the urine in the bladder.

-False positive results may occur in stale urines, in which nitrite has been formed by contamination of the specimen and in
urines containing dyes (derivatives of pyridinium, beetroot).

-Just how to check people, especially children, for urinary tract infections is a subject of much discussion today
     -more than 105 colony-forming units / mL of clean-catch voided urine
     -more than 104 colony-forming units / mL of catheter-obtained voided urine
     -more than 103 colony-forming units / mL of urine obtained by suprapubic aspiration


Leukocyte
-The reaction detects the presence of esterases that occur in granulocytes.

-These enzymes cleave an indoxyl ester and the indoxyl so liberated reacts with a diazonium salt to produce a violet dye.

-Pyuria can be detected even if the urine sample contains damaged or lysed WBC's.


Bilirubin
-Bilirubin metabolism begins with the breakdown of red blood cells (RBCs) by phagocytic cells (cells that consume and
digest other cells).

-Hemoglobin is broken down to heme and globin.

-Heme is converted to bilirubin, which is then carried by albumin in the blood to the liver.

-In the liver, most of the bilirubin is conjugated with glucuronic acid before it is excreted in the bile.
     Conjugated bilirubin is called direct bilirubin
     Unconjugated bilirubin is called indirect bilirubin
     Total bilirubin = DB + IB.

-Conjugated bilirubin is excreted into the bile by the liver and stored in the gall bladder or transferred directly to the small
intestines.

-Bilirubin is further metabolized by bacteria in the intestines to urobilins, which contribute to the color of the feces.

-A small percentage of these compounds are reabsorbed and eventually appear in the urine, where they are referred to as
urobilinogen.

-If the bile ducts are obstructed, direct bilirubin will build up to a high enough level that some of it will escape from the liver
into the blood.
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                                                                                                                               34
-If the levels are high enough, some of it will also appear in the urine.
-Only direct bilirubin appears in the urine.
-Increased direct bilirubin usually means that the biliary (liver secretion) ducts are obstructed.

-Hepatobiliary disease:
     Bilirubinuria generally results when conjugated bilirubin levels in blood are elevated as a result of hepatobiliary disease,
bilirubinuria indicates cholestasis.

-In some cases of hemolytic anemia, bilirubinuria may be secondary to the hemolysis without any evidence of cholestasis.
The renal tubular epithelium is capable of absorbing hemoglobin from the glomerular filtrate and converting it to conjugated
bilirubin, which is then excreted in the urine. This will only occur with intravascular hemolysis, when free hemoglobin is
filtered by the glomerulus.

-The bilirubin pad on the multireagent dipstick detects bilirubin using a specific diazotization reaction and is sensitive to 0.2-
0.4 mg/dL of conjugated bilirubin.

-The color change indicating a positive reaction, however, is a rather subtle transition among shades of beige, and sometimes
is obscured by color inherent in the urine itself (e.g., marked hemoglobinuria).

False negative results
    -large amounts of vitamin C or Nitrite
    -long exposure of the sample to direct light

This test is based on detection of the "peroxidase-like" activity inherent in molecules of heme Hemoglobin and myoglobin
catalyze the oxidation of the indicator by an organic peroxide contained in the test pad
            Hematuria                     Hemoglobinuria                     Myoglobinuria

Mechanism - RBCs lyse on           Mechanism - free Hb filtered     Mechanism - free Mb filtered
contact with the reagent pad,      into urine as a result of        into urine as a result of
causing a positive reaction        hemoglobinemia (usually          myoglobinemia (not visually
(speckled pattern may result if    detectable as visibly red        detectable in plasma).
low-grade)                         plasma)

Clinical - Bleeding into urinary   Clinical - Intravascular         Clinical - Myocyte injury
space; can occur at any level of   hemolysis of any cause:          allowing release of myoglobin
the tract). Commonly due to        immune-mediated, toxic,          which reaches bloodstream
inflammation, trauma,              mechanical, infectious, etc.     and is readily filtered at the
neoplasia, hemostatic disorders                                     glomeruli.


Hematuria: RBC will be present on urine sediment examination (if hematuria is marked, a red precipitate forms after
centrifugation of urine).

Hemoglobinuria: There will be no RBC on the urine sediment and the urine supernatant will be red (remember that RBC
will lyse in very dilute or alkaline urine).

Myoglobinuria: high CK and (maybe) high AST, reflecting muscle injury.• In rare instances, myoglobinuria and
hemoglobinuria can pre-exist in a single patient




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                                                                                                                               35
                                              Urine Microscopy                                                               12/4
Under normal conditions, the urine of healthy people contains a number of erythrocytes, leukocytes and hyaline casts, yet
there is no precise information as to the upper limit of the normal cell range counts in urine excreted daily.

This lack of data is due in part to technical problems and to the wide variation from one individual to another, and in part to
the considerable variability in the percentage of cells and casts that are destroyed and consequently not counted; the extent of
this loss depends on the physicochemical conditions of the urine and the length of time these elements remain in it.

Quantitative assessment is therefore only empirical and approximate; nonetheless, for conventional volumes of 1250 ml of
urine voided per day, it is generally agreed that the approximate, acceptable maximum limits for normal urine are:
    500 erythrocytes/ml
    2000 leukocytes/ml
    > 15 hyaline casts/ml.

Since the various techniques for obtaining total counts are too time-consuming for routine examination, they have not come
into common use and an estimate of the average number of elements in highly magnified microscopic fields (generally 400x)
is preferred.

This is more approximate and even less reliable assessment since it is based on a technique which introduces an additional
number of variables, partly determined by chance (e g. dilution ot the urine) and partly connected with the execution of the
test.

The latter is often be no means standardized: there may be variations in the volume of urine centrifuged, the time and speed
of centrifugation, the volume of the supernatant, the drop of urine examined, etc.

If, however, one does not intend to fix precise limits for normal and abnormal values, or to inaccurate comparisons of the
different examinations, this type of semi-quantative test does provide quite a good practical basis for assessment.

Erythrocytes and leukocytes are not found in the sediment of some healthy people; yet, although opinions differ, one red cell,
one to two leukocytes and only an occasional hyaline cast are generally taken, in a rather arbitrary way, to indicate the upper
normal limit for each high magnified microscopic field observed (400x).

Before drawing any definite conclusion, all borderline results require careful critical assessment, bearing in mind that the
urinary sediment in certain nephropathies may, at least in some stages, be basically normal.
Quantitative or semi-quantitative assessment must be considered as only approximate, especially if successive observations
are being compared.

Erythrocytes
-The morphology of the blood red cells found in urinary sediment is extremely variable.
-There correlations between certain pathological conditions of the kidney and the urinary tract and the morphology of the red
cells in the sediment.
-Using a morphological classification, a first category may include the following elements, typical of hematuria, generally
caused by 'urologic' diseases
-Urinary red cell morphology can be assessed best by Phase-contrast microscopy.
-A second category of red cells in the urinary sediment includes damaged, irregularly shaped cells, showing fractured
membranes, extrusion of cytoplasm, or fragmentation = 'glomerular' red cells
-Extensive fragmentation of the red cells is typical of hematuria in glomerulonephritis and in vascular nephropathies,
proliferative glomerulonephritis or cortical necrosis.
-The few red cells found in the urinary sediment of healthy people are very rarely normal. The red cells from the so-called
'exercise-induced hematuria' are generally abnormal in appearance, which indicates that they have come from the glomerulus
rather than from the bladder

Some elements in the sediment may be mistaken for RBC
   -fungi
   -calcium carbonate crystals
   -small air bubbles.
   -fat droplets
   -small leukocytes
                                                     the guy with the bow tie
                                                                                                                                 36
Leukocytes
Granulocytes, lymphocytes, and monocytes may be found in the urine. However, in routine clinical practice no great interest
is being shown in the accurate identification of these cells. They are taken simply as a non-specific indication of an
inflammatory process in the urinary tract.

Epithelial Cells
Renal tubular epithelial cells, Cells of this kind are found in varying numbers in 90% of glomerular diseases

Epithelial cells from the urinary tract used to be defined as "cells from the upper, middle or lower tracts", according to their
morphology

However, except for the urethra, the excretory system is lined with an epithelium of several layers (transitional or urothelial
epithelium). The characteristics of these layers are common throughout, so that the morphological differences depend on the
layers from which they come and not on the part of the tract where exfoliation took place

Casts
Casts are made up of protein material that has been precipitated into the tubular lumen.
Some of this material is the Tamm-Horsfall urinary protein, produced primarily by the renal tubule in the ascending limb of
the loop of Henle.

This endotubular precipitation is favored by highly concentrated urine with a low pH. With an alkaline pH the hyaline matrix
of the casts either does not form at all or dissolves.

In advanced chronic renal failure, with polyuria and a urinary pH tending to be alkaline, the number of casts decreases,
irrespective of the extent of the damage to the parenchyma.

It is also possible for there to be a large number of casts in the renal parenchyma, and yet a very low number of casts in the
urine. This happens in chronic pyelonephritis and in other interstitial nephropathies in which these elements are blocked in
the kidney and only a small number are passed out in the urine
On the basis of their structure, casts can be classified as:
      -hyaline
      -cellular (red blood cell, epithelial or granulocytic)
      -granular
      -waxy
      -mixed and with inclusions

1) Hyaline casts
The transitory presence of small delicate hyaline casts in urine has no specific significance; casts of this kind can occasionally
be found in the urinary sediment of healthy people under normal conditions, and they are frequently found, even in healthy
people, after strenuous physical exercise or acute dehydration. Hyaline casts are neither specific nor differential for kidney
disease.

-Hyaline casts increase in the following disorders:
             -heart failure
             -Hyperthermia
             -all nephropathies, with or without proteinuria.
             -the presence of cells or fat droplets always indicates some renal damage.

2) Granular casts
-Granular casts may be made up of granules of very varied diameter; these granules are believed to be mainly derived from
cellular debris and tend to become progressively more homogeneous. Granular casts are more specific that hyaline casts.

-Casts of small diameter and with small granules can be found in rapidly reversible pathological conditions (e.g. febrile
diseases with increased catabolism) and, very rarely, also in healthy people. For these reasons, no precise pathological
significance can be attributed to the occasional finding of a few such granular casts

-In contrast, however, persistent granular casts, particularly if the granules are large, always have a pathological significance

                                                     the guy with the bow tie
                                                                                                                                37
3) Hemoglobin cast
-Hemoglobin casts appear in acute hemolysis, transfusion of incompatible blood.
-acquired hemolytic disorders (DIC) inherited hemolytic diseases (glucose-6-phosphate dehydrogenase deficiency)

4) Red cell casts
-Glomerular diseases, in the active stages, are the most frequent cause of red cell casts in the urine, but they can be found in
all the nephropathies that cause hematuria.

5) Waxy casts
-Considered to be the final transformation of all kinds of cast: cellular, granular and hyaline and they always point to severe
renal impairment in at least one area.
-Their significance as markers of severe damage is still greater if they are of medium size or large diameter.

6) Epithelial casts
-These casts are made up of desquamated tubular cells, included in a protein matrix
-indicate active or acute renal disorders
-glomerulonephritis, in interstitial nephritis, in 'acute tubular necrosis'

Crystals
    -crystals usually only form in extreme urine pH
1) Uric acid crystals
    -Uric acid crystals are characteristic of acidic urine
    -their repeated appearance demonstrates the existence of a habitually low urinary pH, which may create a predisposition
         to precipitation in vivo
    -Calcium oxalate are a characteristic of acid urine (calcium phosphate in an alkaline urine)
    -The presence is generally due to urine having been kept for some time at room temperature, or at low temperature
    -Calcium oxalate crystals may be observed in urine of healthy people, especially after they have eaten food rich in oxalic
         acid, such as spinach or cocoa (calcium oxalate is seen in vegetarian diet)
    -Generally there is no connection between this crystalluria and urinary calculi, but if the crystals are large or clustered, or
         if they appear very frequently in the freshly voided urine examined immediately at body temperature, they should be
    considered an abnormal finding.
2) Phosphate crystals
    -Only the presence of magnesium ammonium phosphate in freshly voided urine is now regarded as significant.
    -This finding suggests infection with urease-producing bacteria
3) Cystine crystals
    -The presence of cystine crystals in the sediment is always a very important diagnostic feature, as they are a definite sign
         of urolithiasis or a predisposition to it.




                                                      the guy with the bow tie

				
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