MUST to KNOW in Hematology

Hematology EDTA (Lavende to!"

Modi+ed Westegen ES, (-la. to! t)/e" 0itate (Lig1t /l)e to! t)/e" 5olyyt1emi !atients O%alate

He!ain

Ode o6 Da7 (Heny 89st Edition"

Ode o6 Da7 (Syinge met1od" EDTA ontaining t)/es

S.in !)nt)e

MUST TO KNOW IN HEMATOLOGY  Greek: -Haima = Blood -Logos = Study/science Chelates calcium Invesion# $% Anticoagulant of choice for hematology cell counts and cell morphology Blood smear: prepare w/in 2 hrs referre referred d anticoagulant for platelet count: = !n some patients w/ "#$A "#$A anticoagulated %lood & platelet satellitism = latelet satellitism: platelets adhere to neutrophils & E'et to a)tomated !latelet o)nt * Deeased ' (emedy: (epeat platelet count using citrate )(odak: latelet count * +,+  "#$A  "#$A = Shrinkage of cells cells = .ct = "S( ot for coagulation tests: = !nhi%its 0%rinogen-throm%in reaction = 1actor  is not sta%le in "#$A 2m3 "#$A 4 5,6m3 SS/Citrate (atio = +:7 )Anticoagulant-to-Blood )Anticoagulant-to-Blood 1or coagulation and platelet studies = reser8es reser8es la%ile factors  and !!! = Bu9ered ,2; )5,+5< citrate Invesion# 234% (atio = +:< )Anticoagulant-to-Blood )Anticoagulant-to-Blood  .ct "*cess Citrate = $> A$$ (emedy: (educe the 8olume of citrate Amount of citrate = ?)+55-.ct@)6<6-.ct * m3 B #ou%le/%alanced o*alate )(atio = 2:: aintained cell structures a, 5otassi)m o%alate )aul-.ellers  )aul-.ellers = shrink cells %, Ammoni)m o%alate )intro%es = swell cells !nacti8ation of throm%in Anticoagulant for osmotic fragility test !n8ersion: -7* Not 6o /lood +lm !e!aation# = #istorts cells = roduces roduces %luish %ackground on (omanowskys (omanowskys stain Not 6o oag)lation = !nhi%its throm%in and all stages of coagulation "8acuated tu%e: +, Sterile %lood culture tu%e 2, Citrate )%lue , onadditi8e tu%e )red 7, .eparin )green 6, "#$A )la8ender )la8ende r D, 1luoride )gray +, "#$A 2, Ether anticoagulated tu%es , onadditi8e tu%e 3a8ender ink hite (oyal %lue  $an  $an +, :ingeti!s 8; Ealo/e: less admi*ture w/ tissue Fuice> less pain> less free


0ommon ga)ge (needle" 0ommon lengt1 o6 needle 0olo oded 1)/ (ga)ge"

Angle To)ni>)et -5 )' as to)ni>)et ,eass)e t1e !atient 5osition t1e !atient I< line

Hemato!oiesis Meso/lasti !eiod

He!ati !eiod

Myeloid !eiod

Ad)lts Neonates Mao7 s!eimens

ner8e endings , Lateal !otion o6 t1e !lanta s)6ae o6 t1e 6oot: H+ year old #i9erence from 8enous specimen:  BCs  .g%> .ct> (BCs> platelet eins in the arms )antecu%ital region: +, Median )/ital = preferred> preferred> most sta%le 2, 0e!1ali )lateral ; -asili )medial +<> 25> 2+ (outine: (outine: 25g 25 g +-+,6 inches

9$ = pink 89 = green 88 = gray 82 = %lue/light %lue/turIuoise 8== orange enipuncture: +65 BB: 765  +5-255 once in the skin -7 inches a%o8e the site )J,6-+5cm ot e*ceed +min/2mins rolonged rolonged application hemoconcentration 75-?@ mm.g Crying = cell count 3ying down = hemodilution ) C %y K;> BC 3ying up = hemoconcentration Collect on the other arm !f %oth arms: Stop ! for 2mins = Collect %lood %elow the ! line = Appropriate for all analytes e*cept glucose and phosphorus Cellular formation> proliferation> di9erentiation and maturation of %lood cells 9t1 day o6 gestation  Lolk  Lolk salk = "rythropoiesis "m%ryonic hemoglo%ins: a, Go7e 9 = Meta2 4 "psilon2 %, 5otland = Meta2 4 Gamma2 c, Go7e 8 = Alpha2 4 "psilon2 2d mont1 o6 gestation 1etal 1etal li8er = Granulopoiesis> "rythropoiesis> egakaryopoiesis Spleen> thymus> lymph nodes .emoglo%in production: a; H/: = Alpha2 4 Gamma2 %, H/A9 = Alpha2 4 Beta2 c, H/A8 = Alpha2 4 Gamma2 -et7een =t1 B ?t1 mont1 o6 gestation gestation !esist t1o)g1o)t li6e B = + source s ource of cell production ).ematopoiesis Sternum = principal source of hematopoiesis in adults .%A+ = N<6; .%A2 = +,6-; .%1 = H2; .%1 = D5-K5; .%A = 25-75; +; Te!1ine (0oe" -io!sy = $rephine %iopsy needle )Oamshidi )Oamshi di needle 2, As!iation


0ommon ga)ge (needle" 0ommon lengt1 o6 needle 0olo oded 1)/ (ga)ge"

Angle To)ni>)et -5 )' as to)ni>)et ,eass)e t1e !atient 5osition t1e !atient I< line

Hemato!oiesis Meso/lasti !eiod

He!ati !eiod

Myeloid !eiod

Ad)lts Neonates Mao7 s!eimens

ner8e endings , Lateal !otion o6 t1e !lanta s)6ae o6 t1e 6oot: H+ year old #i9erence from 8enous specimen:  BCs  .g%> .ct> (BCs> platelet eins in the arms )antecu%ital region: +, Median )/ital = preferred> preferred> most sta%le 2, 0e!1ali )lateral ; -asili )medial +<> 25> 2+ (outine: (outine: 25g 25 g +-+,6 inches

9$ = pink 89 = green 88 = gray 82 = %lue/light %lue/turIuoise 8== orange enipuncture: +65 BB: 765  +5-255 once in the skin -7 inches a%o8e the site )J,6-+5cm ot e*ceed +min/2mins rolonged rolonged application hemoconcentration 75-?@ mm.g Crying = cell count 3ying down = hemodilution ) C %y K;> BC 3ying up = hemoconcentration Collect on the other arm !f %oth arms: Stop ! for 2mins = Collect %lood %elow the ! line = Appropriate for all analytes e*cept glucose and phosphorus Cellular formation> proliferation> di9erentiation and maturation of %lood cells 9t1 day o6 gestation  Lolk  Lolk salk = "rythropoiesis "m%ryonic hemoglo%ins: a, Go7e 9 = Meta2 4 "psilon2 %, 5otland = Meta2 4 Gamma2 c, Go7e 8 = Alpha2 4 "psilon2 2d mont1 o6 gestation 1etal 1etal li8er = Granulopoiesis> "rythropoiesis> egakaryopoiesis Spleen> thymus> lymph nodes .emoglo%in production: a; H/: = Alpha2 4 Gamma2 %, H/A9 = Alpha2 4 Beta2 c, H/A8 = Alpha2 4 Gamma2 -et7een =t1 B ?t1 mont1 o6 gestation gestation !esist t1o)g1o)t li6e B = + source s ource of cell production ).ematopoiesis Sternum = principal source of hematopoiesis in adults .%A+ = N<6; .%A2 = +,6-; .%1 = H2; .%1 = D5-K5; .%A = 25-75; +; Te!1ine (0oe" -io!sy = $rephine %iopsy needle )Oamshidi )Oamshi di needle 2, As!iation

= Aspiration needle )Pni8ersity of !llinois sterna needle 5osteio ilia est Safest site for B aspirate/%iopsy umeric e*pression comparing the relati8e num%er of M#E atio granulocytic precursors w/ the relati8e erythroid precursors in the B N< * 8#9 to 4#9 (Ave; 2#9" !nfection = D:+ 3eukemia 3eukemia = 26:+ eutrophilic> "osinophilic> Basophilic precursors = yeloid "rythroid precursors onocytic precursors = not included ercentage ercentage of marro ma rrow w space occupied %y hematopoietic cells -M 0ell)laity compared w/ fat ormocellular marrow marrow )Adult: 5; - J5; hematopoietic cells ' 1at = +5-65; ' .ematopoietic elements = 75-D5; )A8e, 65; 1ats  Yello7  Yello7 -M .ematopoietic cells ,ed -M (ecommended that at least 655> prefera%ly +555 cells %e Mao7 di'eential (ecommended counted for a marrow di9erential di9erential redominant cell )BC in adult B )up to 2; MetamyeloyteC)ve redominant nile gan)loyte H+; cells in B Stem ells Bone forming cells Osteo/lasts Confused w/ plasma cells ater%ug or comet appearance Bone destroying cells Osteolasts Confused w/ megakaryocytes  $ cells 0D8 0D2 B cells 0D9 0D8@ Stem cell marker )lymphoid and myeloid precursor 0D24 Q cells 0D9? 0D=? CA33A )Common A33 Antigen 0D9@ roduced roduced %y the kidney Eyt1o!oietin rimary regulator of erythropoiesis erythropoiesis roduced roduced %y kidney and li8er T1om/o!oietin (egulator (egulator of throm%opoiesis Eyt1o!oiesis + stimulus = .ypo*ia Eyt1o!oiesis ulti-CS1 )Colony Stimulating 1actor 1actor IL32 Stimulates hematopoietic cells roerythro%last/ru%ri%last 9; 5onomo/last /C ratio = K:+ ucleoli = +-2 Can produce up to +D (BCs per + pronormo%last/ru%ri%last pronormo%last/ru%ri%last roru%ricyte 8; -aso!1ili !ntensely %asophilic cytoplasm nomo/last /C ratio = D:+ ucleoli usually not 8isi%le

2; 5oly1omato!1ili nomo/last 4; Ot1o1omi nomo/last =; ,eti)loyte

(u%ricyte Blue-gray to pink-gray cytoplasm 3ast stage capa%le of mitosis +st: .g% synthesis )+st: CB (eticulocyte: (eticulocyte: 3ast /C ratio = 7:+ etaru%ricyte Small pyknotic nucleus )dark> small> nonfunctional /C ratio = +:2 olychromatophilic olychromatophilic erythrocyte/#i9usely %asophilic erythrocyte (omanowsky stain = olychromasia

Supra8ital stain = )4 1ine 1ine reticulum of (A #iscocyte ?; Mat)e ,-0 D-K Rm in diameter 3ife span: +25 days B: ronormo%last (eticulocyte 23= days B: (eticulocyte (BC 938 days Geneal 0ell Mat)ation 01aateistis 6o Le).oytes Immat)e 0ells Mat)e 0ells 3arger Smaller )4 ucleoli )- ucleoli Chromati Chromatin: n: 0ne and delicat delicate e )most )most relia% relia%le le Chromati Chromatin: n: coarse coarse and and clumped clumped )most )most relia%le ucleus: large and round ucleus: round, lo%ulated or segmented Cyto ytopla plasm: da dark %l %lue/%asop sophil hilic )( )(A Cytoplasm: li light %lue )( )(A )- Granules )4 Granules  :  :C ratio :C ratio Gan)lo!oiesis eutrophils Gan)lo!oiesis "osinophils Basophils Blast ature granulocyte 94 days "arliest recognia%le stage in granulocytic series 9; Myelo/last /C ratio = 7:+ ucleoli = 2-6 +st: rimary granules 8; 5omyeloyte /C ratio = 2:+ to :+ ucleoli = 2-  Loungest  Loungest cell in the series series wherein a granulocyte granulocyte can %e 2; Myeloyte identi0ed a, eutrophil myelocyte = rose pink granules %, "osinophil myelocyte = orange-red o range-red granules c, Basophil myelocyte = dark purple or %lue-%lack granules 3ast stage capa%le of mitosis +st: Secondary granules /C ratio = +:+ )- ucleoli  Ou8enile granulocyte 4; Metamyeloyte ot capa%le of mitosis )post-mitotic pool !ndented/kidney-shaped nucleus redominant BC in B Sta%/Sta9  =; -and  Loungest  Loungest cell in the series series present in the peripheral peripheral %lood )normal B = 5-D; or 5-J; C or S shaped nucleus Cur8ed or Sausage shaped nucleus (esem%les elger-.uet cells = . cells: coarser chromatin than sta% cells (ose-pink (ose-pink granules ?a; Segmented ucleus: 2-6 lo%es ne)to!1il #iurnal 8ariation ) Speci0c granules: a, 3ysoyme %, 3actoferrin c, Collagenase d, lasminogen acti8ator e, Aminopeptidase (eddish-orange (eddish-orange granules ?/; Eosino!1il ucleus: usually 2 lo%es #iurnal 8ariation )AC$.

?; -aso!1il

Mono!oiesis Monoyte

Lym!1o!oiesis Lym!1oyte

T lym!1oytes - lym!1oytes N)ll lym!1oytes 5lasma ells di'eentiation 5lasma ell

T1om/o!oiesis 9; Mega.ayo/last 8; 5omega.ayoyte 2; Gan)la mega.ayoyte 4; Mat)e mega.ayoyte

Speci0c granules: ?3arger a, aFor %asic protein %, Acid hydrolase c, Cathepsin d, "osinophil cationic protein d, "osinophil-deri8ed neuroto*in e, "osinophil protein T f, hospholipase ?Smaller a, Arylsulfatase %, Acid phosphatase #ark purple to %lue-%lack granules )water-solu%le ucleus: generally unsegmented or %ilo%ed )rare:  or 7 Speci0c granules: a, .istamine %, .eparin c, "osinophilic chemotactic factor A Mono!oiesis +, ono%last 2, romonocyte , onocyte 3argest cell in B +7-25 Rm in diameter Blue-gray cytoplasm any aurophilic granules )ground glass appearance ucleus: kidney/horse-shoe shaped> may %e folded )%rainlike Lym!1o!oiesis +, 3ympho%last 2, rolymphocyte , 3ymphocyte Small = K-+5Rm )Sie = (BC edium = +5-+2Rm 3arge = +2-+DRm )(are Cytoplasm: %luish )(o%ins egg %lue ucleus: compact D5-K5; 3ong-li8ed )7-+5 years +5-25; Short-li8ed )-7 days Can di9erentiate into plasma cell or memory B cells 3arge granular lymphocyte +5; +, lasma%last 2, roplasmacyte , lasmacyte/lasma cell 3arge well-de0ned hof/perinuclear halo )light staining area in the cytoplasm near the nucleus "ccentric nucleus #eeply %asophilic cytoplasm )(ed/pink cytoplasm: 1lame cell ?A%normal Chromatin: UCart wheel patternV T1om/o!oiesis 6 days )egakaryo%last latelets /C ratio = +5:+ ucleus: may show slight lo%ulation )"ndomitosis /C ratio = 7:+ to J:+ 3argest cell in B Cytoplasm contains coarse clumps of granules aggregating into little %undles> which %ud o9 from the periphery to %ecome

=; Metamega.ayoyt e ?; 5lateletCT1om/oy te

8C2 (?F" 9C2 (22" Endomitosis 8@@@34@@@ 9 1eme mole)le 9 1emoglo/in Mito1ondia 949 amino aids 94? amino aids 01omosome 99 01omosome 9? O%y1emoglo/in Dissoiation 0)ve S1i6t to t1e le6t (OD0"

S1i6t to t1e ig1t (OD0"

Heme synt1esis

platelets ultiple nuclei /C ratio = H+:+ #isintegrated cell surrounded %y platelet

+-7Rm in diameter 3ight %lue to purple ery granular a, Chromomere: granular and centrally located %, .yalomere: surrounds the chromomere> nongranular and clear to light %lue 3ife span: K-++ days Circulating platelets latelets stored in the spleen uclear di8ision w/o cytoplasmic di8ision W of platelets a megakaryocyte can produce + mol E2 7 mol E2 "arly and late heme synthesis Alpha Beta> Gamma> #elta> "psilon> Meta Alpha> Meta Beta> Gamma> #elta> "psilon ormal = Sigmoid in shape T-a*is = .g% concentration in g/d3  L-a*is = E#  CE2  $emperature  2>-#G  p.  AXnity of .g% for E2  .%1 CE2  $emperature  2>-#G  p.  AXnity of .g% for E2

S)inyl oenyme A  Glyine  5yido%al 5O4 ↓ )A3A synthase ↓

D3Aminolev)lini aid ↓

)A3A dehydrase ↓ 5o!1o/ilinogen ↓ )Proporphyrinogen synthase )Proporphyrinogen cosynthase ↓ Uo!o!1yinogen ↓ )Proporphyrinogen decar%o*ylase ↓ 0o!o!o!1yinogen ↓ )Coproporphyrinogen o*idase ↓ 5oto!o!1yinogen ↓

Ateial /lood
Met1emoglo/inC Hemiglo/in (Hi"

S)l61emoglo/in

Eyt1oyte mem/ane

Em/den3Meye1o' !at17ay He%ose mono!1os!1ate s1)nt (5entose 5O4 !at17ay"

)rotoporphyrinogen o*idase ↓ 5oto!o!1yin IJ  :e8 ↓ )1errocheletase ↓ HEME E2 saturation = <6; pE2 = <6 mm.g E2 saturation = J5; pE2 = 75 mm.g pE2 = 2D,D mm.g  p. = .g% aXnity for E2 --- )3  p. = .g% aXnity for E2 --- )( Arterial %lood Blood: Bright red color enous %lood Blood: urplish red color Blood: Cherry red color (e8ersi%le Cannot %ind and carry E2 CE has 255* or 2+5* greater aXnity for .g% compared to E2 Smoking> gas> etc, Blood: Chocolate %rown color (e8ersi%le 1e24 1e4 ethemoglo%in reductase de0ciency "*posure to chemicals/drugs "*posure to nitrite> chlorine and nitrate .% )A%normal hemoglo%in Blood: au8e la8ender !rre8ersi%le Cannot transport E2 Can com%ine w/ CE forming car%o*ysulfhemoglo%in i*ture of o*idied> partially denatured forms of hemoglo%in that form during o*idati8e hemolysis green hemochrome Sulfonamides> aromatic amine drugs Bacteremia: Clostridium perfringens "nterogenous cyanosis 65; protein: +, !ntegral protein: Glycophorin A = Contains sialic acid: )- charge = Meta potential: red cells repel each other 2, eripheral protein: Spectrin and Actin = (esponsi%le for %iconca8e shape of red cells = A%n, Spectrin: .ereditary Spherocytosis> .ereditary "lliptocytosis 75; lipid: +, hospholipids 2, Cholesterol = 3CA$ +5; C.E aFor pathway <5; glycolysis )anaero%ic Glucose 3actic acid = 2 A$ Q de0ciency: common enyme de0ciency +5; glycolysis )aero%ic ro8ides reduced glutathione to pre8ent .g% denaturation GD# de0ciency: most common red cell enyme

,a!o!ot3L)e/eing !at17ay Met1emoglo/in ed)tase !at17ay 5itting 0)lling ,-0 lysis E%tavas)la 1emolysis

Intavas)la 1emolysis

Anisoytosis ,DW Aniso1omia M0< M0H M0H0 Nomoyti nomo1omi anemia Mioyti 1y!o1omi anemia

de0ciency = )4 .ein %odies: denatured .g% )Supra8ital stain: (.. = )4 Bite cells: due to pitting Generates 2>-#G that regulates .g% aXnity for E 2 aintains .g% iron in ferrous )1e 24 state to %e functional (emo8al of inclusion %y the spleen (emo8al of senescent/aged (BCs %y the spleen (BC age = "nymes> A$> Sie> #ensity +; of (BCs/day %roken down %y the mononuclear phagocytic system )S <5; aged red cell destruction w/in the ("S when C is not/incompletely acti8ated  PnconFugated %iliru%in  Prine and fecal uro%ilinogen +5; aged red cell destruction w/in the %lood 8essels when C is completely acti8ated  .aptoglo%in  .emope*in )4 .emoglo%inemia ariation in sie umerical e*pression that correlates w/ the degree of anisocytosis  = ++,6-+7,6; ariation in .g% content )"*, #imorphic anemia  = K5-+55f3 )ormocytic HK5f3 = icrocytic Y+55f3 = acrocytic  = 2J-2 pg  = +-D; )ormochromic H+; = .ypochromic YD; = .yperchromic +, Acute %lood loss 2, .emolytic anemia , Aplastic anemia +, Chronic %lood loss = most common cause 2, $halassemia = -(# , !#A = !ron> $!BC )"*, .ookworm infections   (# 7, Sidero%lastic anemia 6, Chronic disease +, egalo%lastic anemia = itamin B +2/1olate de0ciency 2, onmegalo%lastic anemia = 3i8er disease> alcoholism Congenital = 1anconis anemia AcIuired = radiation> chemical )%enene> drugs )chloramphenicol ancytopenia = BCs> (BCs> (etics lts #i9erentiates !#A )$!BC from other microcytic> hypochromic anemia ormal = Area of palor +/ of the cell diameter +4 = Area of palor +/2 of the cell diameter 24 = Area of palor 2/ of the cell diameter 4 = Area of palor /7 of the cell diameter 74 = $hin rim of hemoglo%in E8al macrocytes .owell-Oolly %odies .ypersegmented neutrophils 

Maoyti nomo1omi anemia A!lasti anemia

TI-0 Degee o6 Hy!o1omia

Megalo/lasti anemia


:oli aid (
S!1eoytosis Mioyti 1y!o1omi 5oi.iloytosis Maoytes Oval maoytes Maoovaloytes Aant1oyte S!) ell T1on ell E1inoyte -) ell Sea )1in ell 0enated ,-0s

0odoyte Taget ell Me%ian 1at ell

Le!toyte S!1eoyte -all -one ell

AIHA vs; HS Stomatoyte

!ne9ecti8e erythropoiesis )pancytopenia ' w/ CS pro%lems +, ernicious anemia = #e0cient in intrinsic factor )produced %y parietal cells for B+2 a%sorption 2, D. latum infection , egetarian diet 7, ala%sorption syndrome = Steatorrhea> sprue ' w/o CS pro%lems +, regnancy 2, #ietary de0ciency , Steatorrhea> sprue (eticulocytosis isi%le on rights stain Blue-gray coloration> pink cytoplasm !ndicates young (BCs  "rythropoietic acti8ity a, .emorrhage %, .emolysis "E1$ "E1$ ariation in shape "S( = o rouleau* formation egalocytes: it, B+2 or folic acid de0ciency Asynchronous de8elopment: mature cytoplasm %ut immature nucleus !rregular spikes/spicules A%normal 3:S ratio ' A%etalipoproteinemia 3i8er diseases "chinocyte: artifactual Burr cell: pathologic (BCs w/ proFections of eIual length and distri%ution  A$ ' "*posure to hypertonic solution ' Artifact in air drying ' Anemia associated w/ renal insuXciency Greek: Qodon = %ell Central hemoglo%inied area )%ulls eye Scanning ": Bell/tall hat shaped  Surface mem%rane to 8olume ratio  Cholesterol Z phospholipid ' .emoglo%inopathies ' $halassemia ' 3i8er disease> postsplenectomy> !#A  $hin 8ariant of a codocyte Spheroid Bicon8e*  Surface area to 8olume ratio #efect of loss of mem%rane: ' .ereditary spherocytosis = ost-splenectomy: Spherocytes ' .emolytic anemia ' Burns ' Banked %lood stored for a long time A!.A = E1$> C.C> )4 #A$ .S = E1$> C.C> )- #A$ Greek: Stoma = mouth outh> slit-like pallor area

Elli!toyte

Ovaloyte

S1istoyte S1ioyte :agmentoyte

Keatoyte Knioytes 5in1 ells Dayoyte Teado!s Mios!1eoyte 5yo!oi.iloyte

Semil)na /odies Hal63moon ell 0esent ell -)ns De!anoytes Si.le ells Menisoytes Ho7ell3olly /odies -aso!1ili sti!!ling 5)ntate /aso!1ilia

5I0A 0a/ot ings

Bowl-shaped  ermea%ility to a4 )ormal: permea%ility to Q 4 ' .ereditary stomatocytosis ' (hnull disease (od/cigar shaped arrower than o8alocytes #efect in cytoskeleton  em%rane protein %and 7,+ "gglike/o8al-shaped ider than elliptocytes Bipolar arrangement in .g%  Cholesterol ' .ereditary o8alocytosis ' egalo%lastic B ' yelodysplasia 1ragmentation produced %y damage of (BC %y 0%rin> altered 8essel walls> prosthetic heart 8al8es ♫ icroangiopathic hemolytic anemia = resence of 0%rin strands in small %lood 8essels Uclothesline-like e9ectV ♫ Burns ♫ $$ ♫ #!C Schistocyte w/ hornlike proFections  $riangular cells 2 palor areas  $eardrop/pear-shaped SIueeing and fragmentation during splenic passage ♫  ♫ .ypersplenism ♫ Se8ere %urns ♫ .ereditary microspherocytosis ♫ .ereditary pyropoikilocytosis = sensiti8ity to temperature = (BCs fragment at 76C )ormal: 7<C 3arge> pale pink staining ghost of the red cell em%rane remaining after the contents ha8e %een released ♫ alaria Spherocytes Schistocytes icrospherocytes Crescent-shaped cell .olly-leaf  olymeriation of deo*ygenated .g% ♫ Sickle cell anemia ♫ Sickle cell disease uclear remnants of #A )from karyorrhe*is 1eulgen )4 ♫ egalo%lastic anemia recipitation of ri%osomes and (A +, 1ine stippling = polychromatophilia ) production of (BCs 2, Course stippling = 3ead poisoning ♫ yrimidine-6-nucleotidase de0ciency = Basophilic stippling !n children = 3ead poisoning !n adults = !#A 1igure of K (emnant of microtu%ules of mitotic spindle ♫ egalo%lastic anemia

Hein /odies

H/H inl)sions H/00 ystals

H/S0 ystals

,inged Sideo/last

Sideoyte

5a!!en1eime /odies

Malaia

Babesia

Aggl)tination ,o)lea)%

Do! o6 NSS Hemoglo/ino!at1ies H/S H/0

recipitated> denatured .g% ultiple .ein %odies itted golf %all appearance (eIuires Supra8ital stain )(.. ♫ GD# de0ciency ♫ Pnsta%le hemoglo%ins ).%. ♫ 1a8ism )1a8a %eans ♫ #rug-induced Acetylphenylhydraine/phenylhydraine = !nduce .ein %odies formation  $etramer of %eta glo%in chains )[7 (eIuires Supra8ital stain )(.. Bar of Gold Clam shell appearance .e*agonal w/ %lunt ends and stain darkly  Solu%ility ashington monument shape #ark-hued crystal of condensed .g% distorts the (BC mem%rane  Solu%ility ucleated (BC )immature that contains nonheme iron particles (eIuires russian %lue stain ♫ Sidero%lastic anemia )!ron o8erload ♫ #S on-nucleated (BC )mature containing iron granules )hemosiderin ♫ Sidero%lastic anemia ♫ #S !ron granules 8isi%le w/ rights stain (esem%les %asophilic stippling = B: eriphery = BS: .omogeneous Pnused iron deposits ♫ Sidero%lastic anemia ♫ #S E$: Anopheles mosIuito %ite aturation stages: (ings Y $rophooite Y Schionts Y Gametocytes P. vivax  = orldwide P. falciparum = hilippines (esistant to alaria: = 1y)a-%-: African = Sickle cell anemia = GD# de0ciency E$: $ick %ite Ualtese crossV (esem%le P. falciparum rings Cold agglutinin disease )anti-! rimary atypical pneumonia )anti-! (BCs in stack of coins arrangement  plasma glo%ulin ♫ ultiple myeloma: proliferation of !g-producing plasma cells )"S( ♫ acroglo%ulinemia #isperses rouleau* formation  $rue agglutination remains intact Hemoglo/ino!at1ies \ualitati8e defect in .g% "*, Su%stitution ]2[2D Glu al ]2[2D Glu 3ys

]2[22D Glu 3ys K5/<5-+55; .%S  .%1 Z .%A2 66-D5; .%A+ 75-76; .%S Si.le ell tait T1alassemia \uantitati8e defect in .g% T1alassemia Alpha-glo%in chain is or a%sent 3t1alassemia a, Adult = [7 = .%. )deletion of /7 alpha genes %, eonate = ^7 = .% Barts )deletion of 7/7 alpha genes Beta-glo%in chain is or a%sent 3t1alassemia .%1 and .%A2  $halassemia maFor 0ooleys anemia .omoygous [-thalassemia  $halassemia minor 0ooleys tait .eteroygous [-thalassemia Alkaline p.: K,D 0ell)lose aetate Hg/ igration: )Cathode Y Anode eleto!1oesis 0  S  :  A9  -ats  I  H " # E G A2 3epore ormal: .%A+ is the fastest )most anodal A%normal: .%. is the fastest )most anodal Acid p.: D,5-D, 0itate aga Hg/ igration: )Cathode Y Anode eleto!1oesis 1 Y A Erigin E Y S Y C " # G +, Sodium meta%isul0te = )4 Sickling of cells Seening test 6o H/S 2, Solu%ility test = Sodium thiosul0te = )4 $ur%idity  in [-thalassemia H/A8 \uantitation: Anion e*change microchromatography Alkali resistant H/: )4 .iC  $ests: +, Alkali denaturation test = .%1 resists alkali denaturation a, Betke )aE. %, Singer )QE. 2, Acid elution test = .%1 resists acid-elution = Cells w/ .%1 = deep pink color = Cells w/ -.%1 = ghost cells +, .eat precipitation test: _65C for 2 hrs Tests 6o )nsta/le Hg/ 2, !sopropanol precipitation test: +J; solution (H/H" Sam!le 0iteia 6o Eyt1oyte Mo!1ology Eval)ation Mo!1ology 01aateistis 7Cin 9 8 2 4 Nomal (!e (!e (!e OIO" (!e Limits OIO" OIO" OIO" (OIO" 5-6 6-+5 +5-25 25-65 Y65 Maoytes ( m" 5-6 6-+5 +5-25 25-65 Y65 Mioytes (P m" 5-2 -+5 +5-65 65-J6 YJ6 Hy!o1omia 5-2 -+5 +5-25 25-65 Y65 5oi.iloytosis -+-6 D-+5 Y+5 -5oly1omato!1ilia -Agg, of   Agg, of 6- umerous -,o)lea)% -7 +5 (BCs aggregate

H/E Si.le ell anemia

5elge3H)et

Hy!esegmentation

(BCs s N)lea A/nomalities .yposegmentation )neutrophil Bilo%ed nucleus: #um%-%ell shaped/spectacle/peanutshaped/Vince-neV (esem%les Sta% cell )$o di9erentiate: . cell has more clumped chromatin ♫ elger-.uet anomaly = Autosomal #ominant ♫ seudo-elger-.uet = AcIuired in myeloproliferati8e disorders N D lo%es )neutrophil A%normal #A synthesis ♫ Pndrit anomaly = hereditary hypersegmentation ♫ egalo%lastic anemia

0yto!lasmi A/nomalities 3arge purple-%lack coarse cytoplasmic granules Alde3,eilly gan)les Accumulation of degraded mucopolysaccharides )all leukocytes ♫ Alder-(eilly anomaly = Autosomal (ecessi8e ♫ ucopolysaccharidoses: .urler> .unter> San0lippo syndrome (esem%le to*ic granules )!$ 3arge purple to %lack granules resem%ling A3( granules To%i gan)les ♫ !nfections ♫ $o*ic states !nfections To%i va)oles  $o*ic states ink or red rod shaped structures A)e ods 1used primary granules )pero*idase positi8e yeloid and monocytic series only w/ mass of Auer rods :aggot ells  )A3 = associated w/ #!C Giant red> %lue to grayish round inclusions )large 01edia.3Higas1i gan)les lysosomal granules Seen in lymphocyte> neutrophil and monocyte 3ysosomal defects latelets lack dense granules ♫ Chediak-.igashi syndrome = Autosomal (ecessi8e )Al%inism ale %lue inclusions deri8ed from (A May3Hegglin inl)sion ♫ ay-.egglin anomaly = Autosomal (ecessi8e = Giant platelets = $hrom%ocytopenia (esem%le #ohle %odies )!$ Single or multiple %lue inclusions Do1le /odies Aggregates of free ri%osomes of rough "( Do1le3Amato /odies (esem%les ♫ !nfections ♫ $o*ic states #ohle %odies IT# In6etions To%i states  $o*ic granules  $o*ic 8acuoles A/nomalities in :)ntion ormal random acti8ity  o/s syndome A%normal chemotactic acti8ity A%normal random and chemotactic acti8ity Lay le).oyte syndome !na%ility of phagocytes to kill ingested microorganisms 01oni Gan)lomato)s

Disease (0GD"

LE ell

Tat ell

!mpaired A#. o*idase !mpaired o*idati8e meta%olism/respiratory %urst  $est: B$ dye test 0ells E%1i/iting 51agoytosis eutrophil w/ large purple homogeneous round inclusion Belie8e to %e a neutrophil that ingested another neutrophil Bu9y coat Smooth and e8enly stained ♫ S3" onocyte w/ ingested lymphocyte (ough and une8enly stained

A/nomalities Involving Lym!1oytes a, $ype ! ,eative lym!1oyte = $urks irritation cell Aty!ial lym!1oyte = lasmacytoid lymphocyte w/ large %lock of chromatin Stim)lated lym!1oyte %, $ype !!
!ntranuclear protein inclusions 5latelet A/nomalities (Mo!1ologi" Giant !latelet ♫ Bernard-Soulier syndrome ♫ ay-.egglin anomaly SmallCmiomega.ayoyte ♫ yelodysplastic syndromes Lage mega.ayoyte Monon)lea mega.ayoyte uncontrolled proliferation and accumulation Le).emia of one or more of the hematopoietic cells Symptoms: 1e8er> weight loss> sweating` hepatosplenomegaly> enlarged lymph nodes )chronic leukemia  B( #ays to D months A)te le).emia redominantly immature cells )%lasts and UproV stages 2 to D months S)/a)te le).emia aria%le 01oni le).emia inimum of + or 2 years redominantly mature cells BC Y+6>555/R3 Le).emi le).emia BC H+6>555/R3 S)/le).emi le).emia )4 A%normal and immature cells in B BC H+6>555/R3 Ale).emi le).emia )- A%normal and immature cells in B #i8ides acute leukemias into lympho%lastic and :en13Ameian3-itis1 mono%lastic (:A-" 0lassi+ation o6 = Su%di8ided according to cellular morphology> A)te Le).emias cytochemical staining results> cytogenetic studies and $ Z B lymphocytes marker results ormocytic> normochromic (BCs A)te le).emia 1AB = N5; %lasts .enry: .E )ow the standard for diagnosis = N25; A)te le).emia in 1ilden K5; A33 25; A33 +, E: yelopero*idase Tests to di'eentiate ALL = )4 A3 6om ANLL = )- A33 2, SBB: Sudan Black B = )4 A3 = )- A33 , $d$: $erminal #eo*yri%onucleotidyltransferase = arker for immature lymphocyte = )4 A33 = )- A33 A)te Lym!1o/lasti Le).emias (ALL" 3ympho%lasts are small and homogeneous )8ary little in L9 sie Childhood A33 3ympho%lasts are large and heterogeneous )8ary in L8 sie Adult A33 Burkitt-type L2 (are 3ympho%lasts are large %ut homogeneous> and 8acuolated A)te Nonlym!1oyti Le).emias (ANLL" Acute myelo%lastic leukemia w/o maturation )A3 w/o M9

D)t1es /odies

mat B: Y5; %lasts H+5; granulocytic cells Acute myelo%lastic leukemia w/ maturation )A3 w/ M8 mat B: Y5; %lasts Y+5; granulocytic cells Acute promyelocytic leukemia )A3 M2 Y5; %lasts Y+5; granulocytic cells Y5; or Y65; promyelocytes )4 1aggot cells = Associated w/ #!C Acute myelomonocytic leukemia )A3 M4 aegelis leukemia 25; to HK5; monocytic cells Acute mono%lastic leukemia w/o maturation M=a Schillings leukemia YK5; monocytic cells )YK5; mono%lasts Acute mono%lastic leukemia w/ maturation M=/ YK5; monocytic cells )HK5; mono%lasts "rythroleukemia M? "rythremic myelosis #i Guglielmos syndrome Y5; %lasts Y65; erythrocytic precursors Acute megakaryocytic leukemia MF Y5; %lasts Y5; megakaryocytic cells 01oni Myelo!oli6eative Disodes roliferation of a%normal pluripotential stem cell M5D Stem cell di9erentiates into the granulocytic )myeloid stem cell> megakaryocytic and erythroid cell lines )4 hiladelphia chromosome: t)< 4`22- - %oth long arms 9; 01oni Myelogeno)s !f )- h chromosome = poor prognosis Le).emia (0ML" Similar to leukomoid reaction> to di9erentiate: a, Chromosome studies %, 3A = ) in 3eukomoid reaction> in C3 8; Myelo+/osis 7C myeloid 1i%rosis and granulocytic hyperplasia of B> w/ granulocytic and megakaryocytic proliferation in the meta!lasia (MMM" li8er and spleen )e*tramedullary )4 #acryocytes  3A B aspirate = impossi%le )dry tap B %iopsy = appropriate  $hrom%ocytosis: +>555 * +5 BCs> lts  3A )Ether polycythemia: -3A 5olyyt1emia Ether names: olycythemia era> olycythemia 9 A/sol)te !olyyt1emia (u%ra8era> aIue Esler disease> anmyelosis  (BC mass ) .ct (BCs> BCs> latelets  "rythropoietin )"E !n response to hypo*ia 8 A/sol)te !olyyt1emia !n patients w/ pulmonary/cardiac disease 7C a!!o!iate !od)tion (BCs> BCs> latelets o6 E5O

 "E !n patients w/ tumors of kidney> li8er> %rain> adrenal and 8 A/sol)te !olyyt1emia pituitary gland 7C ina!!o!iate  (BCs> -BCs> -latelets !od)tion o6 E5O  "E Spurious/Gais%ck polycythemia ,elative !olyyt1emia Associated w/ stress and an*iety -(BC mass  .ct %ecause of decreased plasma 8olume #i9erentiate a%solute from relati8e polycythemia ,-0 mass  (BC mass = A%solute polycythemia -(BC mass = (elati8e polycythemia Myelodys!lasti SyndomeCDysmyelo!oieti Syndome Clonal a%normalities in hematopoietic cells MDS Ure-leukemiaV: can progress to A33 if not treated H5; %last ; Blasts in B ; Blasts in B Comments Di'eentiation H+; H6; ildest type ,e6atoy anemia (,A" H+; H6; (S: (inged ,A 7C inged sideo/last Y+6; (S sidero%last (,A,S" H6; 6-25; ,A 7C e%ess /last (,AE-" Y6; 25-5; ,AE- in tans6omation (,AE-t" H6; 6-25; ersistent 01oni Myelomonoyti monocytosis Le).emia (0MML" Lym!1o!oli6eative disodes roliferation of cells deri8ed from lymphoid stem cell L5D  $/B cells -9; TC- ell le).emia alignancy in8ol8ing lymphoid tissue 8; Lym!1oma a, on-.odgkins lymphoma = proliferation of neoplastic lymphocytes = (appaport classi0cation %, .odgkins lymphoma = proliferation of cells reacting to neoplasm = )4 (eed-Stern%erg cell: large cell w/ large nucleoli )Ewls eye ♫ #iagnosis: 3ymph node %iopsy = (ye classi0cation: %ased on histologic appearance of lymph node %iopsy = Ann-Ar%or: staging %ased on the e*tent of tissue in8ol8ed 3eukemic reticuloendotheliosis 2; Haiy ell le).emia Eriginally B cells w/ hairlike proFections )4 $(A: $artrate-resistant acid phosphatase

4; Myosis 6)ngoides and Seay syndome Myelo!eo%idase (M5O"

S)dan -la. - (S--"

eoplastic $ cells Seary cells: w/ cere%riform nucleus )Brainlike S!eial Stains arker for primary granules and Auer rods )4 A3 )- A33 Stain should only %e done on fresh specimens arker for phospholipids and lipids )4 A3 )- A33 Can %e done on stored specimens arallels E for interpretation

Teminal deo%yi/on)leotidyl Tans6ease (TdT" 5eiodi Aid3S1i' (5AS"

Na!1t1ol AS3D 01looaetate estease

3Na!1t1yl Aetate estease

3Na!1t1yl -)tyate estease Aid !1os!1atase

Tatate esistant aid !1os!1atase (T,A5" Le).oyte al.aline !1os!1atase (LA5"C Ne)to!1il AL5 (NA5"

Tol)idine /l)e

0oo!e B 0)i.s1an.  Nito/l)e tetaoli)m test

#A polymerase immunopero*idase arker for immature lymphocytes )4 A33 )- A3 arker for glycogen> glycoproteins> mucoproteins and . C.E a, ormal: all %lood cells are AS )4 e*cept erythro%last %, A%normal: erythro%lasts are AS )4 = D )4 3+ and 32 = %locklike positi8ity )- 3 )4 D Speci0c esterase arker for mature and immature neutrophils and mast cells )4 A3 )- Ao3 ?6 )- A33 3asts for months onspeci0c esterase arker for monocytes> megakaryocytes and plasma cells )4 Ao3 ?6 = inhi%ited %y buoride )4 Aeg3 ?J )- A3 onspeci0c esterase !denti0es monocytes> promonocytes and mono%lasts )4 Ao3 ?6 = inhi%ited %y buoride )- A3 resent in all hematopoietic cells and found in lysosomes )4 .airy cell leukemia = $(A )4 )4 $ cell leukemia )- on-$ cell leukemia Cannot %e stored "*cellent marker for hairy cell leukemia eutrophil is the only leukocyte that contains this acti8ity 1ound in  granules )etamyelocytes  3eukomoid reaction> > >  rd trimester of pregnancy  C3> CG3> . Should %e done on fresh specimen ♫ Qaplow count: = Count +55 neutrophils = Grade 5 to 74  = 5-+K6 3A Score: 5 = o stain +4 = 1aint stain 24 = ale stain 4 = Strong stain 74 = #eep )ery intense stain Binds w/ acid mucopolysaccharides in %lood cells Strongly metachromatic Pseful for recognition of mast cells and tissue %asophils )metachromatic granules  CG3: Chronic granulocytic leukemia Stain for %asophils (eagent: $oluidine %lue  $est for CG#

(Oiginal"

Modi+ed N-T

:e)lgen eation 5)ssian /l)e stain S)!avital stain (,HH"

-0-

B$: Colorless to pale yellow ---)$o*ic E2 molecules---Y )4 Blue formaan  $est: ♫ .eparinied %lood Centrifuge Bu9y coat )BC 4 B$ )4 1ormaan  = +5; formaan )4 CG# = 5 to negligi%le !nfection = J5; w/ stimulating agent  $est: ♫ Bu9y coat )BC 4 Bacterial suspension ---)B$---Y )4 1ormaan  = K5-+55; formaan CG# = H65; formaan Speci0c reaction for #A .owell-Oolly %odies = 1eulgen )4 Stain for hemosiderin )4 Sidero%last )4 Siderocytes (.. = CB (eticulum = ew methylene %lue .ein %odies = Crystal 8iolet .emoglo%in . = Brilliant cresyl %lue Stain for automated cell counter

HEMOSTASIS 5imay 1emostasis !n8ol8es %lood 8essels and platelets 1ormation of platelet plug  $est: Bleeding time 1unctions: 5latelets Adhesion Acti8ation (elease Aggregation !n8ol8es the coagulation factors Seonday 1ormation of sta%le 0%rin clot 1emostasis  $est: Clotting time Carry %lood from the heart to the capillaries Ateies 5imay Hemostasis (eturn %lood from the capillaries to the heart
5latelet Ad1esion

-enad3So)lie syndome (Giant !latelet syndome"
,elease disodes

K; car%ohydrate arious minerals> water> nucleotides 9; 5ei!1eal one = responsi%le for adhesion and aggregation a, Glycocaly* = outer surface %, lasma mem%rane = consists of 5 or more glycoprotein c, Su%mem%ranous area 8; Sol3gel one = platelet shape Z contractile elements a, icro0laments: actin Z myosin )actomyosin/throm%ostenin = responsi%le for clot retraction %, icrotu%ules = consists of tu%ulin: maintain the platelet shape 2; Oganelle one = alpha Z dense granules = mitochondria> lysosomal granules 4; Mem/ano)s system a, #ense tu%ular system = site of arachidonic acid meta%olism %, Epen canalicular system )surface connecting system = release of granules latelet adherence to e*posed su%endothelial surface )collagen Eccurs in the presence of 8on ille%rand factor !n 8i8o: collagen !n 8itro: glass )- gp!% = receptor for 81

)- 81 = for platelet adhesion orphologic and functional changes in platelets Agonists: su%stance that initiate acti8ation Arachidonic acid ---)Cycloo*ygenase---Y $hrom%o*ane A 2 asoconstrictor Stimulate platelet secretion !nhi%its CET  %leeding time (elease of granules a, Alpha granules = latelet factor = latelet deri8ed growth factor = 1i%rinogen = 1actor  = 81 = [-throm%oglo%ulin = $hrom%ospondin = 1i%ronectin = Al%umin %, #ense granules )CAAS = Calcium = A# = yrophosphate = A$ = Serotonin Storage pool defects: a, Alpha-granule de0ciency ' Gray platelet syndrome ' \ue%ec platelet disorder = )- 1actor  %inding protein )multimerin %, #ense granule de0ciencies ' .ermansky-udlak ' Chediak-.igashi ' iskott-Aldrich syndrome )] Z dense granule deficiency

Im!otant S)/stanes Seeted /y 5latelets B T1ei ,ole in Hemostasis .Q )] 5omote 1i%rinogen )] oag)lation 1actor  )] 1actor !!!:81 )] A# )d 5omote Calcium )d aggegation latelet factor 7 )]  $hrom%ospondin )] Serotonin )d 5omote  $hrom%o*ane A2 precursors )m%,3 vasoonstition latelet-deri8ed growth factor )] = promotes smooth muscle 5omote vas)la growth e!ai [-throm%oglo%ulin )] = chemotactic for fi%ro%lasts lasminogen )] Ot1e systems ]2-antiplasmin )] a'eted C+ esterase inhi%itor )] latelet attachment to each other 5latelet (eIuires 0%rinogen and Ca 24 aggegation )- gp!!%/!!!a comple*: receptor for 0%rinogen Glanmanns t1om/ast1enia inpoint hemorrhagic spots 5ete1iae .emorrhage of %lood into small areas of skin 5)!)a Blood escapes into large areas of skin E1ymosis ose%leed E!ista%is 3eakage of %lood into Foint ca8ities Hemat1osis omiting of %lood Hematemesis Swelling or tumor in the tissues or a %ody ca8ity that contains Hematoma clotted %lood (BC in urine Hemat)ia .g% in urine Hemoglo/in)ia Stool containing dark red or %lack %lood Melena "*cessi8e menstrual %leeding Meno1agia !$  latelet seIuestration %y the spleen = splenomegaly #ilution of platelet count = $hrom%ocytopenia ] W of units transfused

T1om/oytosis

5latelet ad1esion

A>)ied

5latelet o)nt

Uno!ette

5latelet estimate

 Pnits transfused = $hrom%ocytopenia ultiple transfusion: stored %lood contains non8ia%le platelets (eacti8e = moderate increase> asymptomatic )after hemorrhage> splenectomy Autonomous = marked increase> associated w/ throm%otic/hemorrhagic complications )"*, "$: platelet function is a%normal

Q)alitative 5latelet Disodes +, 8# = )- 81 ' latelet aggregation test: = ormal: "pinephrine> Collagen> A# = A%normal: (istocetin 2, BSS = )- gp!% )4 Giant platelets ' latelet aggregation test: = ormal: (istocetin = A%normal: "pinephrine> Collagen> A# , Storage pool defects = Gray platelet )]> .> AS> C.S )d Premia: to*ic meta%olites araproteinemias: coating of platelet mem%rane w/ a%normal protein A3: a%normal megakaryocytes # #rugs: Aspirin = inhi%its CET La/oatoy Tests 6o 5imay Hemostasis +, #irect )eu%auer counting cham%er lt, count/mm = W plt * A1 * #C1 * #1 = W plt * + * +5 * 255 = W plt * 2>555 )(BC pipette = +:255 dilution ' + platelet uncounted = -2>555 plt/mm a, (eese-"cker - Sodium citrate - 1ormaldehyde - Brilliant cresyl %lue %, Guy and 3eake - Sodium o*alate - 1ormaldehyde - Crystal 8iolet c, Brecker-Cronkite - +; ammonium o*alate )phase contrast microscopy ----------------------------------------------------------------------------------------------------d, Pnopette 2, !ndirect )smear latelet count = )W of plts @ +555 (BC * (BC count a, #ameshek %, 1onios c, Elefs #iluent: +; Ammonium o*alate Stand moist cham%er for +6-25mins to allow platelets to settle +,555 ' + platelet uncounted = -+>555 plt/mm K-25 platelets/E!E

1actor: 25>555 (Wedge smea" W-0 estimate (H5:" 1actor: 2>555

5latelet Estimate o6 @34@@@CL =@@@@39@@@@@CL 9@@@@@39=@@@@CL 9=@@@@39@@@CL 8@@@@@34@@@@@CL 4@9@@@3=@@@CL ?@@@@@3F@@@CL $@@@@@L N35lt o)nt -T  5lt o)nt N3-T 5lt o)nt -T 5latelet aggegation test

5latelet ad1esiveness (Salmann"

0lot etation time

0a!illay esistane test To)i>)et test ,)m!el3Leedes test

-leeding time

0oag)lation 6atos

5latelet Estimates (Smea" ,e!ot 5latelet Estimate as arkedly decreased oderately decreased Slightly decreased 3ow normal ormal Slightly increased oderately increased arkedly increased \ualitati8e platelet a%normality rimary 8ascular a%normality 8# Autoimmune throm%ocytopenia Simultaneous Iuantitati8e and Iualitati8e platelet de0ciency Aggregating agents: a, "pinephrine %, Collagen c, A# d, (istocetin Sample: Citrated %lood Centrifuge at D5-+55g for +5mins ( 4 Agonist E,#, monitored )Aggregometer #etermination of in 8itro platelet adhesion lt ct, + = routine collection method lt ct, 2 = collected through glass %ead collecting system ; A = ?)C+-C2 @ C+ * +55  = 2D-D5;  in 8# roportional to platelet count Clot retraction: function of throm%osthenin a, Castor oil/.irsch%oeck )4 #impling/droplet like serum on the surface of %lood drop  = +6-76mins %, Stefanini c, ac1arlane C($ = )8ol, serum @ $ * +55  = 77-DJ; easures capillaries to resist pressure Correlates w/ the degree of throm%ocytopenia +55 mm.g for 6 mins After +6-5mins> count petechiae )or halfway %et, systolic Z diastolic pressure for 6 mins 4+ = 5-+5 petechiae )few 42 = +5-25 petechiae )many 4 = 25-65 petechiae )multiple 47 = Y65 petechiae )conbuent  = 5 to occasional ot repeated on the same arm for J days !n 8i8o measure of primary hemostasis a, #uke method = 0ngertip/earlo%e %, !8y method = uses %lood pressure cu9 )75mm.g puncture forearm Seonday Hemostasis All are produced in the li8er e*cept 1actor !!! !n li8er disease> all coagulation factors are e*cept factor !!! comple* 1actor !!! comple*:

a, !!!: Coagulant )!!!:C %, 81: produced %y megakaryocytes and endothelial cells Acti8ated at cold temp, = !! Z T!

0oag)lation :atos N)meal 5e6eed Name Synonyms 1i%rinogen I rothrom%in rethrom%in II  $issue factor $issue throm%oplastin III Calcium I< roaccelerin 3a%ile factor < Accelerator glo%ulin )aCg rocon8ertin Sta%le factor
5ot1om/in go)!

Diseases

Ca24 dependent it, Q independent Completely consumed during coagulation )4 in plasma )- in serum II Al)E.  )4 in plasma )- in serum -T 5T A5TT Sty!ven TT D).et s     

Disease o6 9 1emostasis  :i/inogen de+ieny  5ot1om/in de+ieny  5aa1emo!1ilia  :ato
:ato I II <





   



  





   

   

    

    A%n A%n

Adso/ed 5lasma 4 4 4 4 4 4

:es1 Se)m

Aged Se)m

4 )H25; 4 4 4 4 4 -

4 4 4 4 4 -

Disodes o6 0oag)lation 0a)sing 0lotting :ato De+ienies In1eited 0oag)lo!at1ies A>)ied 0oag)lo!at1y In1eitane 5atten 0oag)lo!at1y Autosomal recessi8e A0%rinogenemia 3i8er disease Autosomal dominant #ys0%rinogenemia #!C 1i%rinolysis Autosomal recessi8e rothrom%in de0ciency 3i8er disease it, Q de0ciency Anticoagulant therapy Autosomal recessi8e Ewrens disease 3i8er disease 3a%ile factor de0ciency #!C

arahemophilia 1actor !! de0ciency


Autosomal recessi8e


T-linked recessi8e Autosomal dominant Autosomal recessi8e

.emophilia A 8# .emophilia B Christmas disease

J

Autosomal recessi8e

1actor T de0ciency

JI

Autosomal recessi8e

JII

Autosomal recessi8e

Pnclear

3i8er disease #!C 1i%rinolysis Autosomal recessi8e 1letcher trait Pnclear 5K  1itgerald trait Pnclear HMWK  Autosomal recessi8e Common inherited coagulation factor de0ciency :ato
JIII

Autosomal recessi8e

.emophilia C (osenthal syndrome =Common in Oewish descent/ Ashkenai  Oews 1actor T!! de0ciency =o %leeding tendency 1actor T!!! de0ciency

1i%rinolysis 3i8er disease it, Q de0ciency Anticoagulant therapy #!C 1i%rinolysis 3i8er disease it, Q de0ciency Anticoagulant therapy 3i8er disease it, Q de0ciency Anticoagulant therapy Pnclear

0lotting time

5ot1om/in time

Ativated !atial t1om/o!lastin time

Tests 6o Seonday Hemostasis easure the period reIuired for free form of %lood to clot after it has %een remo8ed from the %ody a, Capillary %lood method = #rop or slide = Capillary tu%e/#ale and 3aidlaw = Blue capillary tu%e  = 2-7mins %, hole %lood/3ee and hite  = J-+6mins #etect coagulation factor de0ciencies in8ol8ing e*trinsic and common pathway Citrated %lood Centrifuge at 2555g for +5mins   4 $hrom%oplastin-CaCl2 rgt )4 Clot Begin timing for clot formation on the addition of CaCl 2 rgt  = +5-+2 secs #etect coagulation factor de0ciencies in8ol8ing intrinsic and common pathway  4 A$$ rgt: +, Acti8ators: a, icronied silica %, "llagic acid c, Celite d, Qaolin 2, hospholipids: su%stitute for platelets , CaCl2

Sty!ven timeC,)ssel vi!e venom time

T1om/in time

,e!tilase time

D).ets test =M )ea sol)/ility test

0i)lating antioag)lants L)!)s in1i/ito Tilt t)/e met1od :i/omete

51oto3o!tial detetion o6 lot 6omation

:i/inolysis 5lasminogen ativatos

Begin timing for clot formation on the addition of CaCl 2 rgt  = 26-6 secs "ast !ndian 8iper 8enom Vipera russelli: directly acti8ates factor T #etect coagulation factor de0ciencies in8ol8ing common pathway  4 Styp8en rgt: platelin-CaCl 2 rgt  = D-+5 secs rolonged in: a, 1i%rinogen de0ciency %, resence of 1# or 1S c, resence of throm%olytic agent )"*, streptokinase d, resence of heparin  4 $hrom%in-CaCl2 rgt  = +5-+7 secs "nyme found in the 8enom of Bothrops atrox  snake rolonged in: a, 1i%rinogen de0ciency %, resence of 1# or 1S c, resence of throm%olytic agent ot a9ected %y heparin  4 Atro*in Begin timing for clot formation upon the addition of atro*in  = +5-+6 secs 1or factor T!!! de0ciency (gt: 6 Prea Su%stitutes for urea: - +; monochloroacetic acid - 2; acetic acid ormal = Clot is insolu%le to urea )27 hrs 1, T!!! def, = Clot is solu%le to urea )27 hrs rolonged A$$ and $ not corrected Against the phospholipid portion of prothrom%inase comple* )aTa-Ca24-3 Inst)mentation 6o Tests o6 Hemostasis isual detection of 0%rin clot formation anual techniIue "nd point = clot formation "lectromechanical detection of 0%rin clot formation 1i%rin strand formation is detected using a wire loop or hook w/c has %een incorporated into a semi-automated mechanical instrument #etection of 0%rin clot formation depends on in light scattering associated w/ con8ersion of 0%rinogen 0%rin ' Semi-automated instruments: - "lectra J65 and J65A - 1i%rintimer series - 1 <+5 coagulation analyer ' Automated instruments: - Ertho Qoagula% +DS and 75A - Coag-A-ate T2 and TC - 3A "lectra J55 and K55 #igestion of 0%rin clot Eccurs when plasminogen plasmin +, !ntrinsic acti8ators - T!!a - Qallikrein - .Q  2, $issue type

- Prokinase-like A , $herapeutic acti8ators = treatment for throm%oem%oli - Streptokinase - Prokinase - $issue-like A ]2-antiplasmin = aFor inhi%itor In1i/itos o6 ]2-macroglo%ulin +/inolysis  $hrom%ospondin A inhi%itor + and 2 Crosslinked 0%rin )urea insolu%le Degadation o6 ↓ oss3lin.ed +/in )lasmin /y !lasmin ↓ Comple* ##/"  Comple* L#/#L  Comple* LL/#T#   )#-dimer Degadation o6 non3 :i/inogen o osslin.ed +/in Nonosslin.ed +/in ()ea sol)/le" ↓ and +/inogen /y )lasmin !lasmin ↓ :agment J ↓ )lasmin ↓ :agment Y  :agment D ↓   )lasmin ↓ :agment D  :agment D 5imay +/inolysis o 0%rin monomer o 0%rin polymer o #-dimer  lasminogen acti8ators from damaged/malignant cells Con8erts plasminogen plasmin in the a%sence of 0%rin formation ' rostatic carcinoma #!C = uncontrolled> inappropriate formation of 0%rin w/in the Seonday %lood 8essels +/inolysis w/ 0%rin monomer w/ 0%rin polymer w/ #-dimer = most speci0c for #!C ' !nfection: N. meningitidis ' eoplasm ' Snake %ite ' .$(

W1ole /lood lot lysis time (W-0LT" E)glo/)lin lysis time

5otamine s)l6ate

La/oatoy Eval)ation o6 :i/inolysis Clot should remain intact for appro*imately 7K hrs at JC Clot lysis prior to 7K hrs indicates e*cessi8e systemic 0%rinolysis "uglo%ulins: proteins that ppt, when plasma is diluted w/ .2E Z acidi0ed - lasminogen - lasmin - 1i%rinogen - lasminogen acti8ators lasma 4 Acetic acid pt, euglo%ulin "uglo%ulin 4 throm%in Clot euglo%ulin "uglo%ulin #issol8ed in %u9er ormal = o clot lysis )2 hrs  1i%rinolysis = Clot lysis H2 hrs #etects the presence of 0%rin monomers )2-#!C in the plasma

test Et1anol gelation test Late% D3dime assay Test W-0LT E)glo/)lin lot lysis Et1anol gelation 5otamine s)l6ate D3dime He!ain

Wa6ain 0o)main 0o)madin IN,

ISI

lasma 4 rotamine sulfate 4 "$E. )4 gel-like clot )paracoagulation #etects the presence of 0%rin monomers )2-#!C in the plasma lasma 4 aE. )p. 4 "$E. )4 pptn/gel ost speci0c test for #!C

5imay :i/inolysis Seonday :i/inolysis H 7K hrs H 7K hrs H 2 hrs H 2 hrs 4 4 4 !nFected Action: inhi%its throm%in onitoring: A$$ = sensiti8e> method of choice )CA eutralie w/ protamine sulfate Eral A(1: isconsin Alumni (esearch 1oundation Action: it, Q antagonist> inhi%its !!> !!> !T> T onitoring: $ )reported in !( eutralie w/ itamin Q> 11 !nternational normalied ratio !( = )atient $ @ ormal $ !S! ' !( = 2- - re8ents !> em%olism Z throm%osis ' !( = 2,6-,6 - 1or patients w/ mechanical heart 8al8es !nternational Sensiti8ity !nde* )$ rgt $ rgt is cali%rated w/ anchester rgt = from human %rain throm%oplastin

-ig1t+eld mioso!y Oil immesion mioso!y 51ase3ontast mioso!y :l)oesene mioso!y Eleton mioso!y -asi om!onent o6 Standad lig1t mioso!e

Total magni+ation

Hemoglo/in

HEMATOLOGY 5,O0EDU,ES "*amine %lood 0lms "rythrocyte morphology Z leukocyte di9erential 1or manual platelet counts AA and $/B cell studies E%ser8ation of 0ne ultrastructures of cells )+55>555* magni0cation #iopter rings: adFust for focusing di9erences %etween eyes (u%%er eyeguard: adFust for comfort "yepiece tu%e clamp screw: loosen to rotate head (e8erse facing nosepiece: for ease in specimen manipulation (e8ol8ing nosepiece: use to rotate o%Fecti8es E%Fecti8es: lenses w/c form primary image of specimen 1ield diaphragm: aperture diaphragm w/c restricts area of illumination 1ield diaphragm control ring: adFust sie opening of 0eld diaphragm Coarse focus kno%: %rings slide into 8iew 1ine focus kno%: sharpens image 3amp socket: holds light source !nterpupillary distance scale: indicates distance %etween eyes "yepiece: magnify image formed %y o%Fecti8e lens Stage: holds specimen Slide holder: holds slide in place Condenser control ring: adFusts sie opening of condenser Condenser: aperture diaphragm that controls light Condenser centering screws: center the 0eld of 8iew Condenser focus know: focuses light onto slide T/L tra8el kno%s: mo8es slides on stage Brightness control dial: turns microscope on/o9> adFusts light intensity  $ = Ecular * E%Fecti8e )+5)3E: +5 = +55* )+5).E: 75 = 755* )+5)E!E: +55 = +>555*

Hemoglo/in Detemination A>   Strenuous muscular acti8ity  Altitude =  .g%  3ying down (gt: 5,+ .Cl Comparing the %rownish yellow color of solution to standard comparator %lock (gt: 5,+ aE. ot for new%orns ).%1: alkali resistant +g .g% = +,7m3 E2

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









Aid 1ematin Al.ali 1ematin Gasometi (
+g .g% = ,7Jmg 1e24 CuSE7 method )See BB notes anual and automated #ilution = 5,52m3 %lood: 6m3 rgt )+:26+ (eagents:  #ra%kins reagent )Brown container

a, otassium ferricyanide = ferrous ferric %, otassium cyanide = .i .iC c, onionic detergent = lysis of red cells> decreases tur%idity d, Sodium %icar%onate )Erig, #ra%kins = result is read after +6mins e, #ihydrogen potassium phosphate )od, #ra%kins = result is read after  mins Color intensity is measured at 675nm All forms of .g% are measured e*cept Sulf.% E8eranticoagulation does not a9ect result ♫ $ur%idity = 1alse a, .igh BC count: to correct> centrifuge read supernatant %, .%S Z .%C: to correct> dilute +:+ w/ .2E result * 2 c, 3ipemic %lood: prepare patients %lank )pt, plasma 4 .iC rgt  * (BC = .g%  * .g% = .ct Apply only to normocytic> normochromic red cells 

,)le o6 t1ee

Maomet1ods

Winto/e t)/e

Miomet1od (Adam"

Hematoit Detemination 3arge 8olume of %lood +, intro%e Z 3ands%erg = #ou%le o*alate 2, an Allen = Sodium o*alate , .aden = Sodium o*alate 7, Sanford-agath = Sodium o*alate 6, Bray = .eparin Centrifuge: 2555-255g for 5mins 3ayers )Spun .ct: +st: 1atty layer = %arely 8isi%le unless the patient is lipemic 2nd: lasma rd: Bu9y coat )+mm = +5>555 BCs/mm Bottom: acked cells 3ength = ++,6cm Bore = ,5mm Cali%ration = 5-+55 a, 3eft: 5-+55 )"S( %, (ight: +55-5 ).ct Capillary tu%e: 3ength = J,5-J,6cm )J5-J6mm Bore = +mm Anticoagulant: (ed ).eparin o anticoagulant: Blue Centrifuge: +5>555-+6>555g for 6mins  $rapped plasma: plasma that remains in (BC portion after spinning =  in poikilocytosis ♫ Ad8antages: a, Better packing of cells %, 3ess %lood c, 3ess time consumed ♫ 3ayers:  $op: lasma 2nd: latelets rd: 3eukocytes 7th: (etics Z n(BCs 6th: ature (BCs Bottom: Clayseal )7-Dmm .ct = only computed .ct = C * (BC count 

A)tomated met1ods

,-0 0o)nt ,-0s

A>







,-0 dil)ting )ids

,-0 o)nt

!sotonic solutions +, SS 2, ,K; Sodium citrate , #acies or formol citrate 7, .ayems 6, $oissons D, Bethells J, Gowers #ilution )(BC pipette = 5,6:+55 )Blood: #iluent = +:255 (BC/mm = W (BC * A1 * #C1 * #1 = W (BC * 6 * +5 * 255 = W (BC * +5>555

W-0 0o)nt W-0s W-0 Dil)ting )ids

W-0 o)nt :)1s ,osent1al

S!eis Levy

Im!oved Ne)/a)e

,-0 o)nt

W-0 o)nt N)leated ,-0s

:om)la 6o W-0 oetion

A>  .ypotonic solutions: lyse non-nucleated (BCs +, +-; acetic acid 2, +; .Cl , $urks diluting buid: Gentian 8iolet 4 glacial acetic acid )solid at +JC 4 .2E i* =  mins )$o allow lysis of (BCs #ilution )BC pipette = 5,6:+5 )Blood: #iluent = +:25 3eukocytosis = Pse (BC pipette )+:+55 or +:255 BC/mm = W BC * A1 * #C1 * #1

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

0o)nting 01am/e 2 counting areas "ach CA w/ +D +mm2 sIuares #epth = 5,2mm #epth factor = 6 olume = Area * #epth * W CA = +Dmm2 * 5,2mm * 2 = D,7mm olume/counting cham%er = ,2mm 7 counting areas "ach CA w/ +5 +mm2 sIuares #epth = 5,2mm #epth factor = 6 olume = Area * #epth * W CA = +5mm2 * 5,2mm * 7 = Kmm olume/counting cham%er = 2mm 2 primary sIuares "ach + sIuare w/ < +mm2 2 sIuares #epth = 5,+mm #epth factor = +5 olume = Area * #epth * W CA =
5e!aation and Staining 5oed)es 6o t1e -lood Smea

Te1ni>)es

8= @ (2@34@@" 88mm % 88mm 832mm @;8= in1 (9m" @;= in1 -)'y oat smea T1i. /lood smea Met1anol ,omano7s.ys stains

+, Co8er glass smear )"hrlich 2, Co8er glass and slide )Beacom , edge smear/ush/Spreader slide techniIue 7, Spun smear/Spun/Spinners techniIue = Automated: .emaspinner Angle %etween 2 slides SIuare co8erslip #rop of %lood #istance of %lood drop from the frosted edge of the slide Smear terminates near the end of the slide )automated spreader 1or patients w/ BC count of H+ * +5 causes permanent %lindness rights Giemsa = preferred stain for %lood parasites odi0ed rights-Giemsa 3eishman  Oenner ay-Grunwald ----------------------------------------------------------------------------------------------------Contains: = ethylene %lue )or Aure B - o*idied: %asic = "osin: acidic  $ime %lood smears should %e stained

7Cin 832 1o)s o6 s!eimen olletion Blood and %one marrow staining !H ?;$ alarial parasite staining !H F;8  $hick 0lms E%essively /l)e rolonged staining time stain !nadeIuate washing  $oo high alkalinity of stain #iluents tends to cause e*cessi8e %asophilia !nsuXcient staining E%essively !in. rolonged washing time stain ounting co8erslips %efore they are dry  $oo high acidity of the stain Bu9er may cause e*cessi8e acidophilia Blood 0lm is mo8ed from side to side 0oss3setional o enellation met1od BCs are counted from the tail toward the head of the smear Longit)dinal met1od -attlement met1od ear the tail on a horiontal edge: count  consecuti8e horiontal edge 0elds> count 2 0elds towards the center of the smear> count 2 0elds horiontally> count 2 0elds 8ertically to the edge +55 cells = routine W-0 0o)nting 65 cells = if patient BC count H+,5 * +5
Anemi !atients

Ne)to!1ils Lym!1oytes Monoytes Eosino!1ils -aso!1ils Ne)to!1ilia Eosino!1ilia

Lym!1oytosis

Monoytosis

S1i6t to t1e le6t S1i6t to t1e ig1t ,eti)loyte o)nt  ,etis A/sol)te eti)loyte o)nt 0oeted eti)loyte o)nt ,eti)loyte !od)tion inde%CS1i6t oetion Mat)ation time o6 etis in t1e /lood ,5I  2

 $hicker smear: - larger %lood drop - fast spread - increase angle (elati8e = 7J-JJ; A%solute = +,K-J,K * +5555  = 26-J6 * +5
,5I P 8 Mille dis.  Eosino!1il o)nt Eosino!1ilia

Eosino!enia Eosino!1il dil)ting )ids

T1ons test

!nadeIuate response of B to anemia - Aplastic anemia - !ne9ecti8e erythropoiesis )megalo%lastic anemia ; (etics = (etics )A @ ?(BC )B * < * +55  = 65-65 * +5D/3 Allergic reactions arasitic infections Brucellosis 3eukemias .yperadrenalism )Cushings disease Shock Administration of AC$. Composition: a, hlo*ine/eosin/neutral red iodide = stains eosinophils %, ropylene glycol = lyses (BCs c, a2CE = lyses BCs e*cept eosinophils> intensi0es staining of granules d, .eparin = pre8ents clumping #iluting buids: +, ilots = phlo*ine 2, anners = phlo*ine , (andolphs = phlo*ine 7, .inkleman = eosin 6, $annens = neutral red iodide Assess adrenocortical function Sample +: fasting specimen Sample 2: 7 hrs after administration of AC$. ) eo, count ormal: "o, count: + Y 2 )lower %y 65; A%normal: "o, count: + = 2 )hypoadrenalism 

,-0 indies M0< M0H M0H0 De6etive enti6)ge M0H0 2$ does not o) M0H0 7ill not 6all P88 ES,  ES,



Eyt1oyte Indies (Winto/e Indies" Classify anemia according to (BC morphology C = ).ct @ (BC * +5  = K5-+55 f3 )old: Rm C. = ).g% @ (BC * +5  = 2J-2 pg )old: RRg (arely used C.C = ).g% @ .ct * +55  = +-D; )+-D g/d3 alues a9ected: = .ct = C = C.C !ncorrect calculation )4 cold agglutinins 3ipemia )4 .%S Z .%C Eyt1oyte Sedimentation ,ate (ES," onspeci0c measurement used to detect Z monitor an inbammatory response to tissue inFury "rythrocytes: acrocytes Anemia lasma composition: most important determinant 1i%rinogen ]+-glo%ulin ]2-glo%ulin [-glo%ulin ^-glo%ulin Cholesterol

 $echnical factor: * $ilting =  5 angle = 5; error   $emp, "rythrocytes: icrocytes oikilocytes olycythemia Anisocytes lasma factor: most important determinant Al%umin 3ecithin  $echnical factor: E8eranticoagulation = "#$A = shrinkage of (BC = "S( +5 mins = +, !nitial rouleau* 75 mins = 2, (apid settling of (BCs +5 mins = , 1inal sedimentation of (BCs D5 mins = $otal (eIuires smaller amount of %lood !n8ol8es no dilution 3ength: ++,6cm )++6mm !nternal %ore: ,5mm Anticoagulant: #ou%le o*alate ost sensiti8e (eIuires more %lood 3ength: 55mm !nternal %ore: 2,D6  5,+6mm Anticoagulant: Citrate )%lack Anticoagulant-to-Blood ratio = +:7 Anticoagulant: 2m3 "#$A 4 5,6m3 SS/Citrate 



 ES,

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.ct>

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

Stages o6 ES,

Winto/e B Lands/eg

StandadCOiginal Westegen

Modi+ed Westegen eta Sedimentation ,atio (S," Eyt1oyte Osmoti :agility test (GiVn and San6od met1od" Aso/ate yanide seening

G?5D )oesent seening 5ao%ysmal not)nal 1emoglo/in)ia (5NH" S)ose 1emolysis test

ot a9ected %y anemia aFor disad8antage: reIuires special capillary tu%es and Metafuge MS( = );.ct @ ;Metacrit * +55 Anticoagulant: .eparin ; aCl = W drops aCl * 5,52 Add (BCs> stand for 2hrs at room temp Check for hemolysis )pink/red supernatant : - !nitial hemolysis = tu%e 2+ or 22 )5,72-5,77; - Complete hemolysis = tu%e +D or +J )5,2-5,7; #etects de0ciencies in the pentose phosphate pathway: - GD# - glutathione pero*idase - glutathione reductase (gts: - a ascor%ate - a cyanide ormal = red )- "nyme = %rown GD 4 A# ---)(BC: GD#---Y D-phosphogluconate 4 A#. )buorescence ormal: a* buorescence at +5mins GD# def: 3ittle or no buorescence AcIuired disorder in w/c red cells are a%normally sensiti8e to complement )- #A1 Screening test for . atient (BCs 4 ABE compati%le serum 4 sucrose solution

Hams aidi+ed se)m test

5atient 1as eeived nomal ,-0s 50H

ormal = )- .emolysis . = )4 .emolysis Con0rmatory test for .  $u%e +: atient (BCs 4 normal serum 4 weak acid )5,2 .Cl  $u%e 2: atient (BCs 4 patient serum 4 weak acid )5,2 .Cl  $u%e : atient (BCs 4 normal inacti8ated serum 4 weak acid )5,2 .Cl ormal = )- .emolysis on all tu%es . = )4 .emolysis e*cept on $u%e  )inacti8ated serum atient w/ . 4 %lood transfusion ---).ams test---Y .emolysis 

!gG autoanti- = %iphasic hemolysin - Cold = attaches to (BCs - arm = (BC lysis Donat13Landsteine  $est for C. Ctrl: atient B incu%ate at JC for 5mins incu%ate at JC test for 5mins  $est: atient B incu%ate at 7C for 5mins incu%ate at JC for 5mins ormal = )- hemolysis on test and control C. = )- hemolysis on control %ut )4 hemolysis on test sample A)to1emolysis test Blood alone ---)7K hrs---Y .emolysis: Y5,2 to 2; Blood 4 glucose ---Y .emolysis: 5-5,K; Blood 4 A# ---Y .emolysis: 5-5,<; ----------------------------------------------------------------------------------------------------GD# de0ciency ) = corrects w/ glucose only Q de0ciency )" = corrects w/ A# only ., Spherocytosis = corrects w/ A# and glucose

5otential 0a)ses o6 Eoneo)s ,es)lts 7it1 A)tomated 0ell 0o)ntes 5aamete 0a)ses o6 S!)io)s 0a)ses o6 S!)io)s Inease Deease Cryoglo%ulin Clotting W-0 o)nt Cryo0%rinogen Smudge cells .eparin Premia plus onoclonal proteins immunosuppressants ucleated (BCs latelet clumping Pnlysed (BCs Cryoglo%ulin Clotting 5latelet o)nt Cryo0%rinogen Giant platelets .emolysis .eparin icrocytic (BCs latelet clumping (BC inclusions latelet satellitism BC fragments Cryoglo%ulin Autoagglutination ,-0 o)nt Cryo0%rinogen Clotting Giant platelets .emolysis BC Y65>555/R3 icrocytic (BCs .%CE Y+5; Clotting Hemoglo/in Cryoglo%ulin Sulfhemoglo%in Cryo0%rinogen .emolysis .eparin BC Y65>555/R3 .yper%iliru%inemia 3ipemia onoclonal proteins Cryoglo%ulin Autoagglutination Hematoit

(a)tomated"

Hematoit (mio1t"

Cryo0%rinogen Giant platelets BC Y65>555/R3 .yperglycemia YD55mg/d3 .yponatremia lasma trapping

Clotting .emolysis icrocytic (BCs "*cess "#$A .emolysis .ypernatremia Cryoglo%ulin Cryo0%rinogen Giant platelets .emolysis icrocytic (BCs Swollen (BCs BC Y65>555/R3 Spuriously low .g% Spuriously high .ct

M0<

Autoagglutination BC Y65>555/R3 .yperglycemia (educed red cell deforma%ility

M0H0

Autoagglutination Clotting .emolysis Spuriously high .g% Spuriously low .ct !ncreased: BC count> (BC count> latelet count> .g%> .ct #ecreased: C !ncreased: BC count> .g% #ecreased: latelet count

0yoglo/)lin 0yo+/inogen He!ain 0lotting Hemolysis A)toaggl)tination De+/inated /lood

O!tial lig1t satteing

Eletial im!edane 0o)lte o)nte

Histogams

!ncreased: C.C #ecreased: BC count> (BC count> latelet count> .g%> .ct !ncreased: .g%> C.C> latelet count #ecreased: (BC count> .ct> C !ncreased: C> C.C #ecreased: (BC count> .ct Blood Glass Beads/clips  $ests: UEAAV - E1$ - Autohemolysis test - Acidi0ed serum test

A)tomated 0ell 0o)nte Blood cells when su%Fect to light will create forward Z side light scatters w/c are detected %y photodetector 1orward 3S = cell sie Side 3S/<55/right angle scatter = cell granularity "*, $echnicon autoanalyer Blood cells are nonconductors of electricity, they create impedance or resistance of current when passed in a solution that conduct electricity "*, Sysme* counter> Coulter counter  $riplicate count )* a, Blood is diluted +:D265 )isotonic ♫ (BCs = D-D5f3 ♫ lts = 2-25f3 %, Blood is diluted +:26+ )hypotonic ♫ 3ymphocytes = 6-<5f3 ♫ onocytes = <5-+D5f3 ♫ Granulocytes = +D5-765f3 (BCs> BCs> plts J3a%is - .oriontal/a%scissa - Sie of cells  Y3a%is - ertical/ordinate - um%er of cells

O1ms la7

<*I%, here:  = 8oltage ! = current ( = resistance 5ositive eo  0o)nt# -EAX ♫ Bu%%les ♫ "*traneous electrical pulses ♫ Aperture plug Negative eo  0o)nt ♫ !mproper setting of aperture error ; of (BCs that are polychromatophilic 5oly1omasia gading Slig1t = +; 9 = ; 8 = 6; 2 = +5; 4 = Y++; Nomoyti 9; De6etive 6omation o6 ,-0s o t1e !esene o6 t)mo Nomo1omi ,-0s ells in -M# Aplastic anemia 3eukemia .odgkins disease ultiple myeloma 3eukoerythro%lastosis etastatic cancer Anemia of renal Z endocrine disease Anemia of inbammatory disease 8; A/nomal 1emoglo/in ineased dest)tion o6 ,-0s Certain acIuired hemolytic anemia . Sickle cell anemia .# Anemia of chronic renal insuXciency Hemolyti anemias 9; Intinsi de6ets 7Cin ,-0 a; Heeditay  mem/ane de6ets Spherocytosis "lliptocytosis Acanthocytosis Stomatocytosis (h null disease %, Heeditay  enyme de6ets GD# Q  c, Heeditay & 1emoglo/ino!at1ies Sickle cell disease .emoglo%in C disease d; Unsta/le 1emoglo/in disease .emoglo%in " disease e; Heeditay & defecti8e glo%in synthesis $halassemia f, A>)ied . 8; E%tao!)s)la a)ses: nonimmune acIuired hemolytic anemias Chemicals> to*ins> 8enoms hysical trauma: disorders causing fragmentation )%urns> cardiac replacement 8al8es> A.A> .PS 2; Intao!)s)la a)ses: immune hemolytic anemias !soimmune anti%odies: incompati%le %lood transfusion> .# Autoimmune anti%odies: warm/cold reacting> drug-induced 4; Misellaneo)s 

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